21 CFR 1271.390 - Applicability.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Applicability. 1271.390 Section 1271.390 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND...

21 CFR 1271.330 - Applicability.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Applicability. 1271.330 Section 1271.330 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND...

21 CFR 1271.330 - Applicability.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Applicability. 1271.330 Section 1271.330 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND...

21 CFR 1271.330 - Applicability.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Applicability. 1271.330 Section 1271.330 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND...

21 CFR 1271.390 - Applicability.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Applicability. 1271.390 Section 1271.390 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND...

21 CFR 1271.330 - Applicability.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Applicability. 1271.330 Section 1271.330 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND...

21 CFR 1271.390 - Applicability.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Applicability. 1271.390 Section 1271.390 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND...

21 CFR 1271.190 - Facilities.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Facilities. 1271.190 Section 1271.190 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND...

21 CFR 1271.200 - Equipment.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Equipment. 1271.200 Section 1271.200 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND...

SERPINE2 is a possible candidate promotor for lymph node metastasis in testicular cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nagahara, Akira; Nakayama, Masashi; Oka, Daizo

2010-01-22

Testicular germ cell tumors (TGCTs) commonly metastasize to the lymph node or lung. However, it remains unclear which genes are associated with TGCT metastasis. The aim of this study was to identify gene(s) that promoted human TGCT metastasis. We intraperitoneally administered conditioned medium (CM) from JKT-1, a cell-line from a human testicular seminoma, or JKT-HM, a JKT-1 cell sub-line with high metastatic potential, into mice with JKT-1 xenografts. Administration of CM from JKT-HM significantly promoted lymph node metastasis. A cDNA microarray analysis showed that JKT-HM cells highly expressed the Serpine peptidase inhibitor, clade E, member 2 (SERPINE2), which encodes amore » secreted protein. Administration of CM from SERPINE2-silenced JKT-HM cells inhibited lymph node metastasis in the xenograft model, compared with administration of CM from JKT-HM cells. There was no significant difference in xenograft volume. Moreover, administration of CM from SERPINE2-over-expressing JKT-1 was likely to promote lymph node metastasis in the xenograft model. There was no difference in the in vitro proliferation or migration of JKT-1 cells cultured with CM from JKT-HM cells, compared to that with CM from JKT-1. There was no promotion of proliferation or lymphangiogenesis in the xenografts, as measured by Ki-67 and LYVE-1 immunohistochemistry, respectively. Although we could not clarify how SERPINE2 promoted lymph node metastasis, it may be a promoter in the development of lymph node metastasis in the human seminoma cells in a mouse xenograft model.« less

Suppression of Angiogenesis and Therapy of Human Colon Cancer Liver Metastasis by Systemic Administration of Interferon-α1

PubMed Central

Ozawa, Shutaro; Shinohara, Hisashi; Kanayama, Hiro-omi; Bruns, Christiane J; Bucana, Corazon D; Ellis, Lee M; Davis, Darren W; Fidler, Isaiah J

2001-01-01

Abstract The purpose of this study was to determine whether systemic administration of interferon-alpha (IFN-α) can inhibit liver metastasis produced in nude mice by human colon cancer cells. KM12L4 (IFN-α-sensitive) or KM12L4 IFNR (IFN-α-resistant) cells were injected into the spleen of nude mice. Seven days later, the mice were treated with subcutaneous (s.c.) injections of IFN-α (70,000 units/week) at different dosing schedules (1, 2, or 7 times/week). Significant inhibition of tumor growth, vascularization and expression of basic fibroblast growth factor (bFGF) or matrix metalloproteinase-9 (MMP-9) mRNA and protein occurred in mice given daily injections of IFN-α. Kinetic analysis of therapy showed that daily s.c. administrations of 10,000 units of IFN-α induced apoptosis in liver metastasis-associated endothelial cells, followed by inhibition of tumor cell division and apoptosis of tumor cells. These data suggest that the antiangiogenic activity of IFN-α-2a depends on frequent administration of the optimal biologic dose. PMID:11420751

21 CFR 1271.270 - Records.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Records. 1271.270 Section 1271.270 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND...

21 CFR 1271.270 - Records.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Records. 1271.270 Section 1271.270 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND...

21 CFR 1271.270 - Records.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Records. 1271.270 Section 1271.270 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND...

21 CFR 1271.215 - Recovery.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Recovery. 1271.215 Section 1271.215 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND...

21 CFR 1271.225 - Process changes.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Process changes. 1271.225 Section 1271.225 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES...

21 CFR 1271.260 - Storage.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Storage. 1271.260 Section 1271.260 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND...

Age-dependent metabolic and immunosuppressive effects of Tacrolimus

PubMed Central

Krenzien, Felix; Quante, Markus; Heinbokel, Timm; Seyda, Midas; Minami, Koichiro; Uehara, Hirohito; Biefer, Hector Rodriguez Cetina; Schuitenmaker, Jeroen M.; Gabardi, Steven; Splith, Katrin; Schmelzle, Moritz; Petrides, Athena K.; Azuma, Haruhito; Pratschke, Johann; Li, Xian C.; ElKhal, Abdallah; Tullius, Stefan G.

2016-01-01

Immunosuppression in elderly recipients has been underappreciated in clinical trials. Here, we assessed age-specific effects of the calcineurin inhibitor Tacrolimus (TAC) in a murine transplant model and assessed its clinical relevance on human T-cells. Old recipient mice exhibited prolonged skin graft survival when compared to young animals following TAC administration. More importantly, half of the TAC dose was sufficient in old mice to achieve comparable systemic trough levels. TAC administration was able to reduce pro-inflammatory IFN-γ cytokine production and promote IL-10 production in old CD4+ T-cells. In addition, TAC administration decreased IL-2 secretion in old CD4+ T-cells more effectively while inhibiting the proliferation of CD4+ T-cells in old mice. Both, TAC treated murine and human CD4+ T-cells demonstrated an age-specific suppression of intracellular calcineurin levels and Ca2+-influx, two critical pathways in T-cell activation. Of note, depletion of CD8+ T-cells did not alter allograft survival outcome in old TAC treated mice, suggesting that TAC age-specific effects were mainly CD4+ T-cell mediated. Collectively, our study demonstrates age-specific immunosuppressive capacities of TAC that are CD4+ T-cell mediated. The suppression of calcineurin levels and Ca2+-influx in both, old murine and human T-cells emphasizes on the clinical relevance of age-specific effects when utilizing TAC. PMID:27754593

T lymphocyte recruitment by interleukin-8 (IL-8). IL-8-induced degranulation of neutrophils releases potent chemoattractants for human T lymphocytes both in vitro and in vivo.

PubMed Central

Taub, D D; Anver, M; Oppenheim, J J; Longo, D L; Murphy, W J

1996-01-01

IL-8 has been shown to be a human neutrophil and T cell chemoattractant in vitro. In an effort to assess the in vivo effects of IL-8 on human leukocyte migration, we examined the ability of rhIL-8 to induce human T cell infiltration using a human/mouse model in which SCID mice were administered human peripheral blood lymphocytes intraperitoneally, followed by subcutaneous injections of rhIL-8. rhIL-8 induced predominantly murine neutrophil accumulation by 4 h after administration while recombinant human macrophage inflammatory protein-1beta (rhMIP-1beta) induced both murine monocytes and human T cell infiltration during the same time period as determined by immunohistology. Interestingly, 72 h after chemokine administration, a marked human T cell infiltrate was observed in the IL-8 injection site suggesting that rhIL-8 may be acting indirectly possibly through a murine neutrophil-derived T cell chemoattractant. This hypothesis was confirmed using granulocyte-depleted SCID mice. Moreover, human neutrophils stimulated in vitro with IL-8 were found to release granule-derived factor(s) that induce in vitro T cell and monocyte chemotaxis and chemokinesis. This T cell and monocyte chemotactic activity was detected in extracts of both azurophilic and specific granules. Together, these results demonstrate that neutrophils store and release, upon stimulation with IL-8 or other neutrophil activators, chemoattractants that mediate T cell and monocyte accumulation at sites of inflammation. PMID:8621778

21 CFR 1271.195 - Environmental control and monitoring.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Environmental control and monitoring. 1271.195 Section 1271.195 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS...

21 CFR 1271.210 - Supplies and reagents.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Supplies and reagents. 1271.210 Section 1271.210 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES...

21 CFR 1271.220 - Processing and process controls.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Processing and process controls. 1271.220 Section 1271.220 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS...

Blockade of invariant TCR-CD1d interaction specifically inhibits antibody production against blood group A carbohydrates

PubMed Central

Tazawa, Hirofumi; Irei, Toshimitsu; Tanaka, Yuka; Igarashi, Yuka; Tashiro, Hirotaka

2013-01-01

Previously, we detected B cells expressing receptors for blood group A carbohydrates in the CD11b+CD5+ B-1a subpopulation in mice, similar to that in blood group O or B in humans. In the present study, we demonstrate that CD1d-restricted natural killer T (NKT) cells are required to produce anti-A antibodies (Abs), probably through collaboration with B-1a cells. After immunization of wild-type (WT) mice with human blood group A red blood cells (A-RBCs), interleukin (IL)-5 exclusively and transiently increased and the anti-A Abs were elevated in sera. However, these reactions were not observed in CD1d−/− mice, which lack NKT cells. Administration of anti-mouse CD1d blocking monoclonal Abs (mAb) prior to immunization abolished IL-5 production by NKT cells and anti-A Ab production in WT mice. Administration of anti-IL-5 neutralizing mAb also diminished anti-A Ab production in WT mice, suggesting that IL-5 secreted from NKT cells critically regulates anti-A Ab production by B-1a cells. In nonobese diabetic/severe combined immunodeficient (NOD/SCID/γcnull) mice, into which peripheral blood mononuclear cells from type O human volunteers were engrafted, administration of anti-human CD1d mAb prior to A-RBC immunization completely inhibited anti-A Ab production. Thus, anti-CD1d treatment might constitute a novel approach that could help in evading Ab-mediated rejection in ABO-incompatible transplant recipients. PMID:23943651

Synergistic effect of sequential administration of mitoguazone (MGBG) and gemcitabine in treating tissue cultured human breast cancer cells and mammary rat tumors.

PubMed

Ishmael, D Richard; Chen, Wei R; Nordquist, John A; Liu, Hong; Nordquist, Robert E

2003-04-01

Modulation of cancer chemotherapeutic drugs has been attempted to increase efficacy and overcome resistance to the chemotherapeutic agent. Studies have shown schedule-dependent interactions in combined use of chemotherapeutic drugs. Mitoguazone (MGBG), an old drug with possible modulating activity, was used in combination with gemcitabine, a relatively new cancer drug, in treating tissue cultured human breast cancer cells and mammary rat tumors. Tissue cultured BOT-2 cancer cells were first treated with varying concentrations of gemcitabine and MGBG, independently. Combinations of the two drugs were then used with different scheduled administrations. Marked synergistic activity was found between gemcitabine and MGBG when the MGBG was given first, followed by gemcitabine 24 hours later. A non-toxic dose of MGBG enhanced the toxicity of gemcitabine by eight orders of magnitude using MTT assays in the tissue cultured human breast cancer cell study. The sequential administration of MGBG and gemcitabine also increased the survival rate of rats bearing mammary tumors in our pilot animal study.

Non-Invasive Cell-Based Therapy for Traumatic Optic Neuropathy

DTIC Science & Technology

2015-06-01

Morphological and Functional Changes in an Animal Model of Retinitis Pigmentosa . Vis Neurosci, 2013: 1-13. Bin Lu, Catherine W. Morgans, Sergey Girman...of human retinal progenitor cells for treatment of retinitis pigmentosa 2013, ARVO, A0106. Benjamin Bakondi; YuChun Tsai; Bin Lu; Sergey...Systemic administration of MSCs significantly preserved retinal ganglion cell survival after TON. (d) Systemic administration of MSCs also promote limited

21 CFR 1271.60 - What quarantine and other requirements apply before the donor-eligibility determination is complete?

Code of Federal Regulations, 2011 CFR

2011-04-01

... DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND... must quarantine semen from anonymous donors until the retesting required under § 1271.85(d) is complete...

21 CFR 1271.60 - What quarantine and other requirements apply before the donor-eligibility determination is complete?

Code of Federal Regulations, 2010 CFR

2010-04-01

... DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND... must quarantine semen from anonymous donors until the retesting required under § 1271.85(d) is complete...

Age-Dependent Metabolic and Immunosuppressive Effects of Tacrolimus.

PubMed

Krenzien, F; Quante, M; Heinbokel, T; Seyda, M; Minami, K; Uehara, H; Biefer, H R C; Schuitenmaker, J M; Gabardi, S; Splith, K; Schmelzle, M; Petrides, A K; Azuma, H; Pratschke, J; Li, X C; ElKhal, A; Tullius, S G

2017-05-01

Immunosuppression in elderly recipients has been underappreciated in clinical trials. Here, we assessed age-specific effects of the calcineurin inhibitor tacrolimus (TAC) in a murine transplant model and assessed its clinical relevance on human T cells. Old recipient mice exhibited prolonged skin graft survival compared with young animals after TAC administration. More important, half of the TAC dose was sufficient in old mice to achieve comparable systemic trough levels. TAC administration was able to reduce proinflammatory interferon-γ cytokine production and promote interleukin-10 production in old CD4 + T cells. In addition, TAC administration decreased interleukin-2 secretion in old CD4 + T cells more effectively while inhibiting the proliferation of CD4 + T cells in old mice. Both TAC-treated murine and human CD4 + T cells demonstrated an age-specific suppression of intracellular calcineurin levels and Ca 2+ influx, two critical pathways in T cell activation. Of note, depletion of CD8 + T cells did not alter allograft survival outcome in old TAC-treated mice, suggesting that TAC age-specific effects were mainly CD4 + T cell mediated. Collectively, our study demonstrates age-specific immunosuppressive capacities of TAC that are CD4 + T cell mediated. The suppression of calcineurin levels and Ca 2+ influx in both old murine and human T cells emphasizes the clinical relevance of age-specific effects when using TAC. © 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.

Combined effects of interleukin-7 and stem cell factor administration on lymphopoiesis after murine bone marrow transplantation.

PubMed

Chung, Brile; Min, Dullei; Joo, Lukas W; Krampf, Mark R; Huang, Jing; Yang, Yujun; Shashidhar, Sumana; Brown, Janice; Dudl, Eric P; Weinberg, Kenneth I

2011-01-01

The decreased ability of the thymus to generate T cells after bone marrow transplantation (BMT) is a clinically significant problem. Interleukin (IL)-7 and stem cell factor (SCF) induce proliferation, differentiation, and survival of thymocytes. Although previous studies have shown that administration of recombinant human IL-7 (rhIL-7) after murine and human BMT improves thymopoiesis and immune function, whether administration of SCF exerts similar effects is unclear. To evaluate independent or combinatorial effects of IL-7 and SCF in post-BMT thymopoiesis, bone marrow (BM)-derived mesenchymal stem cells transduced ex vivo with the rhIL-7 or murine SCF (mSCF) genes were cotransplanted with T cell-depleted BM cells into lethally irradiated mice. Although rhIL-7 and mSCF each improved immune reconstitution, the combination treatment had a significantly greater effect than either cytokine alone. Moreover, the combination treatment significantly increased donor-derived common lymphoid progenitors (CLPs) in BM, suggesting that transplanted CLPs expand more rapidly in response to IL-7 and SCF and may promote immune reconstitution. Our findings demonstrate that IL-7 and SCF might be therapeutically useful for enhancing de novo T cell development. Furthermore, combination therapy may allow the administration of lower doses of IL-7, thereby decreasing the likelihood of IL-7-mediated expansion of mature T cells. 2011. Published by Elsevier Inc.

Epratuzumab modulates B-cell signaling without affecting B-cell numbers or B-cell functions in a mouse model with humanized CD22.

PubMed

Özgör, Lamia; Brandl, Carolin; Shock, Anthony; Nitschke, Lars

2016-09-01

Treatment of systemic lupus erythematosus patients with epratuzumab (Emab), a humanized monoclonal antibody targeting CD22, leads to moderately reduced B-cell numbers but does not completely deplete B cells. Emab appears to induce immunomodulation of B cells, but the exact mode of action has not been defined. In the present study, we aimed to understand the effects of Emab on B cells using a humanized mouse model (Huki CD22), in which the B cells express human instead of murine CD22. Emab administration to Huki CD22 mice results in rapid and long-lasting CD22 internalization. There was no influence on B-cell turnover, but B-cell apoptosis ex vivo was increased. Emab administration to Huki CD22 mice had no effect on B-cell numbers in several lymphatic organs, nor in blood. In vitro exposure of B cells from Huki CD22 mice to Emab resulted in decreased B-cell receptor (BCR) induced Ca(2+) mobilization, whereas B-cell proliferation after Toll-like receptor (TLR) stimulation was not affected. In addition, IL-10 production was slightly increased after TLR and anti-CD40 stimulation, whereas IL-6 production was unchanged. In conclusion, Emab appears to inhibit BCR signaling in a CD22-dependent fashion without strong influence on B-cell development and B-cell populations. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Selective accumulation of PpIX and photodynamic effect after aminolevulinic acid treatment of human adenomyosis xenografts in nude mice.

PubMed

Suzuki-Kakisaka, Haruka; Murakami, Takashi; Hirano, Toru; Terada, Yukihiro; Yaegashi, Nobuo; Okamura, Kunihiro

2008-10-01

To evaluate the effect of photodynamic therapy with aminolevulinic acid (ALA) on human adenomyosis xenografts in a mouse model. Human adenomyosis tissues were implanted SC into nude mice. We measured 5-aminolevulinic acid pharmacokinetics in these mice by analyzing tissue sections 1 to 6 hours after intraperitoneal administration. Twenty-four hours after photodynamic therapy, we evaluated tissue morphologic features. Department of obstetrics and gynecology at a university hospital in Japan. Immunodeficient mice. Tissue grafts were taken from women with adenomyosis attending a university hospital. Photodynamic treatment. Peak fluorescence after intraperitoneal ALA administration and tissue histological changes 24 hours after photodynamic therapy. Peak fluorescence was observed 3 hours after intraperitoneal administration. Histological studies revealed decreased numbers of epithelial and stromal cells in adenomyosis models after therapy. Photodynamic therapy with ALA caused extensive cell death in human adenomyosis tissues implanted into nude mice. Photodynamic treatment using ALA is a potential treatment for patients with adenomyosis uteri.

Effect of human umbilical cord blood stem cell transplantation on oval cell response in 2-AAF/CCL4 liver injury model: experimental immunohistochemical study.

PubMed

Abdellatif, Hussein; Shiha, Gamal; Saleh, Dalia M; Eltahry, Huda; Botros, Kamal G

2017-01-01

Oval cells, specific liver progenitors, are activated in response to injury. The human umbilical cord blood (hUCB) is a possible source of transplantable hepatic progenitors and can be used in cases of severe liver injury. We detected the effect of hUCB stem cell transplantation on natural response of oval cells to injury. Twenty-four female albino rats were randomly divided into three groups: (A) control, (B) liver injury with hepatocyte block, and (C) hUCB transplanted group. Hepatocyte block was performed by administration of 2-acetylaminofluorene (2-AAF) for 12 days. CCL4 was administrated at day 5 from experiment start. Animals were sacrificed at 9 days post CCL4 administration, and samples were collected for biochemical and histopathological analysis. Oval cell response to injury was evaluated by the percentage of oval cells in the liver tissue and frequency of cells incorporated into new ducts. Immunohistochemical analysis of oval cell response to injury was performed. There was significant deviation in the hUCB-transplanted (4.9 ± 1.4) and liver injury groups (2.4 ± 0.9) as compared to control (0.89 ± 0.4) 9 days post injury. Detection of oval cell response was dependant on OV-6 immunoreactivity. For mere localization of cells with human origin, CD34 antihuman immunoreactivity was performed. There was no significant difference in endogenous OV-6 immunoreactivity following stem cell transplantation as compared to the liver injury group. In vivo transplantation of cord blood stem cells (hUCB) does not interfere with natural oval cell response to liver injury.

Engraftment of Human Mesenchymal Stem Cells in a Rat Photothrombotic Cerebral Infarction Model : Comparison of Intra-Arterial and Intravenous Infusion Using MRI and Histological Analysis

PubMed Central

Byun, Jun Soo; Kim, Jae Kyun; Jung, Jisung; Ha, Bon Chul; Park, Serah

2013-01-01

Objective This study aimed to evaluate the hypotheses that administration routes [intra-arterial (IA) vs. intravenous (IV)] affect the early stage migration of transplanted human bone marrow-derived mesenchymal stem cells (hBM-MSCs) in acute brain infarction. Methods Male Sprague-Dawley rats (n=40) were subjected to photothrombotic infarction. Three days after photothrombotic infarction, rats were randomly allocated to one of four experimental groups [IA group : n=12, IV group : n=12, superparamagnetic iron oxide (SPIO) group : n=8, control group : n=8]. All groups were subdivided into 1, 6, 24, and 48 hours groups according to time point of sacrifice. Magnetic resonance imaging (MRI) consisting of T2 weighted image (T2WI), T2* weighted image (T2*WI), susceptibility weighted image (SWI), and diffusion weighted image of rat brain were obtained prior to and at 1, 6, 24, and 48 hours post-implantation. After final MRI, rats were sacrificed and grafted cells were analyzed in brain and lung specimen using Prussian blue and immunohistochemical staining. Results Grafted cells appeared as dark signal intensity regions at the peri-lesional zone. In IA group, dark signals in peri-lesional zone were more prominent compared with IV group. SWI showed largest dark signal followed by T2*WI and T2WI in both IA and IV groups. On Prussian blue staining, IA administration showed substantially increased migration and a large number of transplanted hBM-MSCs in the target brain than IV administration. The Prussian blue-positive cells were not detected in SPIO and control groups. Conclusion In a rat photothrombotic model of ischemic stroke, selective IA administration of human mesenchymal stem cells is more effective than IV administration. MRI and histological analyses revealed the time course of cell migration, and the numbers and distribution of hBM-MSCs delivered into the brain. PMID:24527188

Neuroblast survival depends on mature vascular network formation after mouse stroke: role of endothelial and smooth muscle progenitor cell co-administration.

PubMed

Nih, Lina R; Deroide, Nicolas; Leré-Déan, Carole; Lerouet, Dominique; Soustrat, Mathieu; Levy, Bernard I; Silvestre, Jean-Sébastien; Merkulova-Rainon, Tatiana; Pocard, Marc; Margaill, Isabelle; Kubis, Nathalie

2012-04-01

Pro-angiogenic cell-based therapies constitute an interesting and attractive approach to enhancing post-stroke neurogenesis and decreasing neurological deficit. However, most new stroke-induced neurons die during the first few weeks after ischemia, thus impairing total recovery. Although the neovascularization process involves different cell types and various growth factors, most cell therapy protocols are based on the biological effects of single-cell-type populations or on the administration of heterogeneous populations of progenitors, namely human cord blood-derived CD34(+) cells, with scarce vascular progenitor cells. Tight cooperation between endothelial cells and smooth muscle cells/pericytes is critical for the development of functional neovessels. We hypothesized that neuroblast survival in stroke brain depends on mature vascular network formation. In this study, we injected a combination of endothelial progenitor cells (EPCs) and smooth muscle progenitor cells (SMPCs), isolated from human umbilical cord blood, into a murine model of permanent focal ischemia induced by middle cerebral artery occlusion. The co-administration of SMPCs and EPCs induced enhanced angiogenesis and vascular remodeling in the peri-infarct and infarct areas, where vessels exhibited a more mature phenotype. This activation of vessel growth resulted in the maintenance of neurogenesis and neuroblast migration to the peri-ischemic cortex. Our data suggest that a mature vascular network is essential for neuroblast survival after cerebral ischemia, and that co-administration of EPCs and SMPCs may constitute a novel therapeutic strategy for improving the treatment of stroke. © 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

Histological observation of goblet cells following topical rebamipide treatment of the human ocular surface: A case report.

PubMed

Kase, Satoru; Shinohara, Toshiya; Kase, Manabu

2015-02-01

The topical administration of rebamipide (Mucosta®), an antiulcer agent, clinically increases the mucin level of tear film. The aim of this study was to report the histological changes of goblet cells following the topical administration of rebamipide to a patient with nevus of the lacrimal caruncle. A 62-year-old male exhibited a pigmented nodule located in the lacrimal caruncle in the left eye. An excisional biopsy and subsequent surgical resection were conducted at the caruncle, prior to and three months after topical rebamipide administration. Histologically, a biopsy specimen revealed a pigmented nevus beneath the caruncle epithelium containing a few goblet cells [4 cells/high power field (HPF)]. A few nevus cells were present at the surgical margin. By contrast, the secondary resected specimen obtained three months after the initiation of topical rebamipide treatment revealed the epithelium and nevus, where numerous goblet cells were present (28 cells/HPF), and mucin-like substances were markedly secreted from the goblet cells. Topical rebamipide markedly increased the number of goblet cells and stimulated the secretion of mucin-like substances in the caruncular tissue of a human patient. These results suggest that topical rebamipide is useful in patients following surgery and/or biopsy to support tissue repair of the ocular surface.

Effects of human interleukin-18 and interleukin-12 treatment on human lymphocyte engraftment in NOD-scid mouse

PubMed Central

Senpuku, Hidenobu; Asano, Toshihiko; Matin, Khairul; Salam, M Abdus; Tsuha, Yuzo; Horibata, Shigeo; Shimazu, Yoshihito; Soeno, Yuichi; Aoba, Takaaki; Sata, Tetsutaro; Hanada, Nobuhiro; Honda, Mitsuo

2002-01-01

NOD/LtSz-prkdcscid/prkdcscid (non-obese diabetic-severe combine immunodeficiency; NOD-scid) mice grafted with human peripheral blood lymphoid cells have been used as an in vivo humanized mouse model in various studies. However, cytotoxic human T cells are induced in this model during immune responses, which gives misleading results. To assist in grafting of human lymphocytes without the induction of cytotoxic human T cells, we investigated the effects of T helper type 1 (Th1) and Th2 cytokines on human lymphocyte grafting and migration, as well as the production of immunoglobulin deposited in glomeruli and human immunodeficiency virus-1 (HIV-1) infection using NOD-scid mice. Administration of interleukin-18 (IL-18) and IL-12 enhanced the grafting of human CD4+ and CD8+ T cells in the mice, whereas co-administration prevented grafting due to interferon-γ-dependent apoptosis. Immunoglobulin A (IgA) deposits were observed in mice treated with IL-18 alone, but not in those given phosphate-buffered saline, IL-12 alone, or IL-18 + IL-12. A high rate of HIV infection was also observed in the IL-18-treated group. Together, these results indicate that IL-18 may be effective for the grafting and migration of CD4+ and CD8+ T cells, except for the induction of apoptosis and regulation of class-switching IgA. IL-18-administered NOD-scid mice provide a useful small humanized model for the study of HIV infection and IgA nephropathy. PMID:12383203

CD19xCD3 DART protein mediates human B-cell depletion in vivo in humanized BLT mice

PubMed Central

Tsai, Perry; Thayer, William O; Liu, Liqin; Silvestri, Guido; Nordstrom, Jeffrey L; Garcia, J Victor

2016-01-01

Novel therapeutic strategies are needed for the treatment of hematologic malignancies; and bispecific antibody-derived molecules, such as dual-affinity re-targeting (DART) proteins, are being developed to redirect T cells to kill target cells expressing tumor or viral antigens. Here we present our findings of specific and systemic human B-cell depletion by a CD19xCD3 DART protein in humanized BLT mice. Administration of the CD19xCD3 DART protein resulted in a dramatic sustained depletion of human CD19+ B cells from the peripheral blood, as well as a dramatic systemic reduction of human CD19+ B-cell levels in all tissues (bone marrow, spleen, liver, lung) analyzed. When human CD8+ T cells were depleted from the mice, no significant B-cell depletion was observed in response to CD19xCD3 DART protein treatment, confirming that human CD8+ T cells are the primary effector cells in this in vivo model. These studies validate the use of BLT humanized mice for the in vivo evaluation and preclinical development of bispecific molecules that redirect human T cells to selectively deplete target cells. PMID:27119115

78 FR 44575 - Sickle Cell Disease Treatment Demonstration Program

Federal Register 2010, 2011, 2012, 2013, 2014

2013-07-24

... DEPARTMENT OF HEALTH AND HUMAN SERVICES Health Resources and Services Administration Sickle Cell... Extension: Sickle Cell Disease Treatment Demonstration Program (U1E) Awards to Three Currently Funded... the Sickle Cell Disease Treatment Demonstration Program. Three of these awards will end on August 31...

[COMPARISON OF CYTOPROTECTIVE EFFECTS OF HEMANTANE AND AMANTADINE UNDER CONDITIONS OF 6-HYDROXYDOPAMINE NEUROTOXIN ACTION ON CULTURED HUMAN NEUROBLASTOMA CELLS].

PubMed

Logvinov, I O; Antipova, T A; Nepoklonov, A V; Valdman, E A

2016-01-01

Potential neuroprotective activity of the novel antiparkinsonian drug hemantane (hydrochloride N-2-(adamantyl)-hexamethylenimine) in comparison to amantadine has been studied in various regimes of administration on human neuroblastoma SH-SY5Y cell line injury induced by 6-hydroxydopamine (6-OHDA), which is used as in vitro model of dopaminergic neurons for Parkinson's disease. Two regimes of hemantane and amantadine administration in a range of final concentrations 10⁻⁶-10⁻⁸ M were used either prior to or immediately after 6-OHDA introduction. MTT colorimetric assay was used to assess the viability of test cells. Significant decrease in viability of SH-SY5Y cells treated with 6-OHDA was observed. The addition of hemantane to cell medium produced cytoprotective effects in both regimes of administration--before and after 6-OHDA--at concentrations 10⁻⁷ M and 10⁻⁶-10⁻⁸ M, respectively. Amantadine in con- centrations 10⁻⁷-10⁻⁸ M was effective to increase cell survival only when administered after 6-OHDA. These results show that hemantane has a greater neu-roprotective potential in comparison to amantadine.

Multipotent progenitor cells are present in human peripheral blood.

PubMed

Cesselli, Daniela; Beltrami, Antonio Paolo; Rigo, Silvia; Bergamin, Natascha; D'Aurizio, Federica; Verardo, Roberto; Piazza, Silvano; Klaric, Enio; Fanin, Renato; Toffoletto, Barbara; Marzinotto, Stefania; Mariuzzi, Laura; Finato, Nicoletta; Pandolfi, Maura; Leri, Annarosa; Schneider, Claudio; Beltrami, Carlo Alberto; Anversa, Piero

2009-05-22

To determine whether the peripheral blood in humans contains a population of multipotent progenitor cells (MPCs), products of leukapheresis were obtained from healthy donor volunteers following the administration of granulocyte colony-stimulating factor. Small clusters of adherent proliferating cells were collected, and these cells continued to divide up to 40 population doublings without reaching replicative senescence and growth arrest. MPCs were positive for the transcription factors Nanog, Oct3/4, Sox2, c-Myc, and Klf4 and expressed several antigens characteristic of mesenchymal stem cells. However, they were negative for markers of hematopoietic stem/progenitor cells and bone marrow cell lineages. MPCs had a cloning efficiency of approximately 3%, and following their expansion, retained a highly immature phenotype. Under permissive culture conditions, MPCs differentiated into neurons, glial cells, hepatocytes, cardiomyocytes, endothelial cells, and osteoblasts. Moreover, the gene expression profile of MPCs partially overlapped with that of neural and embryonic stem cells, further demonstrating their primitive, uncommitted phenotype. Following subcutaneous transplantation in nonimmunosuppressed mice, MPCs migrated to distant organs and integrated structurally and functionally within the new tissue, acquiring the identity of resident parenchymal cells. In conclusion, undifferentiated cells with properties of embryonic stem cells can be isolated and expanded from human peripheral blood after granulocyte colony-stimulating factor administration. This cell pool may constitute a unique source of autologous cells with critical clinical import.

Tumor-specific delivery of biologics by a novel T-cell line HOZOT

PubMed Central

Onishi, Teppei; Tazawa, Hiroshi; Hashimoto, Yuuri; Takeuchi, Makoto; Otani, Takeshi; Nakamura, Shuji; Sakurai, Fuminori; Mizuguchi, Hiroyuki; Kishimoto, Hiroyuki; Umeda, Yuzo; Shirakawa, Yasuhiro; Urata, Yasuo; Kagawa, Shunsuke; Fujiwara, Toshiyoshi

2016-01-01

“Cell-in-cell” denotes an invasive phenotype in which one cell actively internalizes in another. The novel human T-cell line HOZOT, established from human umbilical cord blood, was shown to penetrate a variety of human cancer cells but not normal cells. Oncolytic viruses are emerging as biological therapies for human cancers; however, efficient viral delivery is limited by a lack of tumor-specific homing and presence of pre-existing or therapy-induced neutralizing antibodies. Here, we report a new, intriguing approach using HOZOT cells to transmit biologics such as oncolytic viruses into human cancer cells by cell-in-cell invasion. HOZOT cells were successfully loaded via human CD46 antigen with an attenuated adenovirus containing the fiber protein of adenovirus serotype 35 (OBP-401/F35), in which the telomerase promoter regulates viral replication. OBP-401/F35–loaded HOZOT cells were efficiently internalized into human cancer cells and exhibited tumor-specific killing by release of viruses, even in the presence of anti-viral neutralizing antibodies. Moreover, intraperitoneal administration of HOZOT cells loaded with OBP-401/F35 significantly suppressed peritoneally disseminated tumor growth in mice. This unique cell-in-cell property provides a platform for selective delivery of biologics into human cancer cells, which has important implications for the treatment of human cancers. PMID:27901098

Induction of autophagy by spermidine promotes longevity.

PubMed

Eisenberg, Tobias; Knauer, Heide; Schauer, Alexandra; Büttner, Sabrina; Ruckenstuhl, Christoph; Carmona-Gutierrez, Didac; Ring, Julia; Schroeder, Sabrina; Magnes, Christoph; Antonacci, Lucia; Fussi, Heike; Deszcz, Luiza; Hartl, Regina; Schraml, Elisabeth; Criollo, Alfredo; Megalou, Evgenia; Weiskopf, Daniela; Laun, Peter; Heeren, Gino; Breitenbach, Michael; Grubeck-Loebenstein, Beatrix; Herker, Eva; Fahrenkrog, Birthe; Fröhlich, Kai-Uwe; Sinner, Frank; Tavernarakis, Nektarios; Minois, Nadege; Kroemer, Guido; Madeo, Frank

2009-11-01

Ageing results from complex genetically and epigenetically programmed processes that are elicited in part by noxious or stressful events that cause programmed cell death. Here, we report that administration of spermidine, a natural polyamine whose intracellular concentration declines during human ageing, markedly extended the lifespan of yeast, flies and worms, and human immune cells. In addition, spermidine administration potently inhibited oxidative stress in ageing mice. In ageing yeast, spermidine treatment triggered epigenetic deacetylation of histone H3 through inhibition of histone acetyltransferases (HAT), suppressing oxidative stress and necrosis. Conversely, depletion of endogenous polyamines led to hyperacetylation, generation of reactive oxygen species, early necrotic death and decreased lifespan. The altered acetylation status of the chromatin led to significant upregulation of various autophagy-related transcripts, triggering autophagy in yeast, flies, worms and human cells. Finally, we found that enhanced autophagy is crucial for polyamine-induced suppression of necrosis and enhanced longevity.

Immunologic "vaccination" for the prevention of autoimmune diabetes (type 1A).

PubMed

Simone, E A; Wegmann, D R; Eisenbarth, G S

1999-03-01

Diabetes type 1A is an autoimmune condition characterized by lymphocytic infiltration of islets and selective destruction of insulin-secreting beta-cells. Numerous investigators have prevented diabetes in animal models with a variety of antigens and routes of administration. It is also now possible to identify high-risk individuals even before the appearance of autoantibodies. These advances have created the opportunity to design and begin human prevention trials. This review focuses on a variety of immunomodulatory approaches (including administration of adjuvants, autoantigens, T-cells, T-cell receptors, and DNA) that we have collectively termed immunologic "vaccination." In addition, we discuss the potential benefits and dangers of these approaches and issues relating to the design of human trials.

Potential therapeutic effect of the secretome from human uterine cervical stem cells against both cancer and stromal cells compared with adipose tissue stem cells.

PubMed

Eiró, Noemí; Sendon-Lago, Juan; Seoane, Samuel; Bermúdez, María A; Lamelas, Maria Luz; Garcia-Caballero, Tomás; Schneider, José; Perez-Fernandez, Roman; Vizoso, Francisco J

2014-11-15

Evidences indicate that tumor development and progression towards a malignant phenotype depend not only on cancer cells themselves, but are also deeply influenced by tumor stroma reactivity. The present study uses mesenchymal stem cells from normal human uterine cervix (hUCESCs), isolated by the minimally invasive method of routine Pap cervical smear, to study their effect on the three main cell types in a tumor: cancer cells, fibroblasts and macrophages. Administration of hUCESCs-conditioned medium (CM) to a highly invasive breast cancer MDA-MB-231 cell line and to human breast tumors with high cell proliferation rates had the effect of reducing cell proliferation, modifying the cell cycle, inducing apoptosis, and decreasing invasion. In a xenograft mouse tumor model, hUCESCs-CM reduced tumor growth and increased overall survival. In cancer-associated fibroblasts, administration of hUCESCs-CM resulted in reduced cell proliferation, greater apoptosis and decreased invasion. In addition, hUCESCs-CM inhibited and reverted macrophage differentiation. The analysis of hUCESCs-CM (fresh and lyophilized) suggests that a complex paracrine signaling network could be implicated in the anti-tumor potential of hUCESCs. In light of their anti-tumor potential, the easy cell isolation method, and the fact that lyophilization of their CM conserves original properties make hUCESCs good candidates for experimental or clinical applications in anticancer therapy.

Redistribution, Hyperproliferation, Activation of Natural Killer Cells and CD8 T Cells, and Cytokine Production During First-in-Human Clinical Trial of Recombinant Human Interleukin-15 in Patients With Cancer

PubMed Central

Conlon, Kevin C.; Lugli, Enrico; Welles, Hugh C.; Rosenberg, Steven A.; Fojo, Antonio Tito; Morris, John C.; Fleisher, Thomas A.; Dubois, Sigrid P.; Perera, Liyanage P.; Stewart, Donn M.; Goldman, Carolyn K.; Bryant, Bonita R.; Decker, Jean M.; Chen, Jing; Worthy, Tat'Yana A.; Figg, William D.; Peer, Cody J.; Sneller, Michael C.; Lane, H. Clifford; Yovandich, Jason L.; Creekmore, Stephen P.; Roederer, Mario; Waldmann, Thomas A.

2015-01-01

Purpose Interleukin-15 (IL-15) has significant potential in cancer immunotherapy as an activator of antitumor CD8 T and natural killer (NK) cells. The primary objectives of this trial were to determine safety, adverse event profile, dose-limiting toxicity, and maximum-tolerated dose of recombinant human IL-15 (rhIL-15) administered as a daily intravenous bolus infusion for 12 consecutive days in patients with metastatic malignancy. Patients and Methods We performed a first in-human trial of Escherichia coli–produced rhIL-15. Bolus infusions of 3.0, 1.0, and 0.3 μg/kg per day of IL-15 were administered for 12 consecutive days to patients with metastatic malignant melanoma or metastatic renal cell cancer. Results Flow cytometry of peripheral blood lymphocytes revealed dramatic efflux of NK and memory CD8 T cells from the circulating blood within minutes of IL-15 administration, followed by influx and hyperproliferation yielding 10-fold expansions of NK cells that ultimately returned to baseline. Up to 50-fold increases of serum levels of multiple inflammatory cytokines were observed. Dose-limiting toxicities observed in patients receiving 3.0 and 1.0 μg/kg per day were grade 3 hypotension, thrombocytopenia, and elevations of ALT and AST, resulting in 0.3 μg/kg per day being determined the maximum-tolerated dose. Indications of activity included clearance of lung lesions in two patients. Conclusion IL-15 could be safely administered to patients with metastatic malignancy. IL-15 administration markedly altered homeostasis of lymphocyte subsets in blood, with NK cells and γδ cells most dramatically affected, followed by CD8 memory T cells. To reduce toxicity and increase efficacy, alternative dosing strategies have been initiated, including continuous intravenous infusions and subcutaneous IL-15 administration. PMID:25403209

21 CFR 864.5240 - Automated blood cell diluting apparatus.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Automated blood cell diluting apparatus. 864.5240 Section 864.5240 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... § 864.5240 Automated blood cell diluting apparatus. (a) Identification. An automated blood cell diluting...

21 CFR 864.5240 - Automated blood cell diluting apparatus.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Automated blood cell diluting apparatus. 864.5240 Section 864.5240 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... § 864.5240 Automated blood cell diluting apparatus. (a) Identification. An automated blood cell diluting...

21 CFR 864.5240 - Automated blood cell diluting apparatus.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Automated blood cell diluting apparatus. 864.5240 Section 864.5240 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... § 864.5240 Automated blood cell diluting apparatus. (a) Identification. An automated blood cell diluting...

Morphological Changes of Human Corneal Endothelial Cells after Rho-Associated Kinase Inhibitor Eye Drop (Ripasudil) Administration: A Prospective Open-Label Clinical Study

PubMed Central

Okumura, Naoki; Suganami, Hideki; Kinoshita, Shigeru

2015-01-01

Purpose To investigate the effect and safety of a selective Rho kinase inhibitor, ripasudil 0.4% eye drops, on corneal endothelial cells of healthy subjects. Design Prospective, interventional case series. Methods In this study, 6 healthy subjects were administered ripasudil 0.4% in the right eye twice daily for 1 week. Morphological changes and corneal endothelial cell density were examined by noncontact and contact specular microscopy. Central corneal thickness and corneal volume of 5 mm-diameter area of center cornea were analyzed by Pentacam Scheimpflug topography. All the above measurements were conducted in both eyes before administration, 1.5 and 6 hours after the initial administration on day 0; and in the same manner after the final administration on day 7. Results By noncontact specular microscopy, indistinct cell borders with pseudo guttae were observed, but by contact specular microscopy, morphological changes of corneal endothelial cells were mild and pseudo guttae was not observed after single and repeated administration of ripasudil in all subjects. These changes resolved prior to the next administration, and corneal endothelial cell density, central corneal thickness and corneal volume were not changed throughout the study period. Conclusion Transient morphological changes of corneal endothelial cells such as indistinct cell borders with pseudo guttae were observed by noncontact specular microscopy in healthy subjects after ripasudil administration. Corneal edema was not observed and corneal endothelial cell density did not decrease after 1 week repetitive administration. These morphological changes were reversible and corneal endothelial cell morphology returned to normal prior to the next administration. Trial Registration JAPIC Clinical Trials Information 142705 PMID:26367375

Functional repair of p53 mutation in colorectal cancer cells using trans-splicing.

PubMed

He, Xingxing; Liao, Jiazhi; Liu, Fang; Yan, Junwei; Yan, Jingjun; Shang, Haitao; Dou, Qian; Chang, Ying; Lin, Jusheng; Song, Yuhu

2015-02-10

Mutation in the p53 gene is arguably the most frequent type of gene-specific alterations in human cancers. Current p53-based gene therapy contains the administration of wt-p53 or the suppression of mutant p53 expression in p53-defective cancer cells. . We hypothesized that trans-splicing could be exploited as a tool for the correction of mutant p53 transcripts in p53-mutated human colorectal cancer (CRC) cells. In this study, the plasmids encoding p53 pre-trans-splicing molecules (PTM) were transfected into human CRC cells carrying p53 mutation. The plasmids carrying p53-PTM repaired mutant p53 transcripts in p53-mutated CRC cells, which resulted in a reduction in mutant p53 transcripts and an induction of wt-p53 simultaneously. Intratumoral administration of adenovirus vectors carrying p53 trans-splicing cassettes suppressed the growth of tumor xenografts. Repair of mutant p53 transcripts by trans-splicing induced cell-cycle arrest and apoptosis in p53-defective colorectal cancer cells in vitro and in vivo. In conclusion, the present study demonstrated for the first time that trans-splicing was exploited as a strategy for the repair of mutant p53 transcripts, which revealed that trans-splicing would be developed as a new therapeutic approach for human colorectal cancers carrying p53 mutation.

21 CFR 801.3 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-04-01

... a surgically or naturally formed cavity of the human body. A device is regarded as an implantable... FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES... use. Human cells, tissues, or cellular or tissue-based product (HCT/P) regulated as a device means an...

Development of an orally-administrative MELK-targeting inhibitor that suppresses the growth of various types of human cancer.

PubMed

Chung, Suyoun; Suzuki, Hanae; Miyamoto, Takashi; Takamatsu, Naofumi; Tatsuguchi, Ayako; Ueda, Koji; Kijima, Kyoko; Nakamura, Yusuke; Matsuo, Yo

2012-12-01

We previously reported MELK (maternal embryonic leucine zipper kinase) as a novel therapeutic target for breast cancer. MELK was also reported to be highly upregulated in multiple types of human cancer. It was implied to play indispensable roles in cancer cell survival and indicated its involvement in the maintenance of tumor-initiating cells. We conducted a high-throughput screening of a compound library followed by structure-activity relationship studies, and successfully obtained a highly potent MELK inhibitor OTSSP167 with IC₅₀ of 0.41 nM. OTSSP167 inhibited the phosphorylation of PSMA1 (proteasome subunit alpha type 1) and DBNL (drebrin-like), which we identified as novel MELK substrates and are important for stem-cell characteristics and invasiveness. The compound suppressed mammosphere formation of breast cancer cells and exhibited significant tumor growth suppression in xenograft studies using breast, lung, prostate, and pancreas cancer cell lines in mice by both intravenous and oral administration. This MELK inhibitor should be a promising compound possibly to suppress the growth of tumor-initiating cells and be applied for treatment of a wide range of human cancer.

Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4+ T-cell responses

PubMed Central

Santana, Vinicius Canato; Almeida, Rafael Ribeiro; Ribeiro, Susan Pereira; Ferreira, Luís Carlos de Souza; Kalil, Jorge; Rosa, Daniela Santoro; Cunha-Neto, Edecio

2015-01-01

T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4+T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4+ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4+ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4+ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4+ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4+ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4+ T-cell immunity. PMID:26602876

MiR-590 Promotes Transdifferentiation of Porcine and Human Fibroblasts Toward a Cardiomyocyte-Like Fate by Directly Repressing Specificity Protein 1.

PubMed

Singh, Vivek P; Mathison, Megumi; Patel, Vivekkumar; Sanagasetti, Deepthi; Gibson, Brian W; Yang, Jianchang; Rosengart, Todd K

2016-11-10

Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells represents a promising potential new therapy for treating heart disease, inducing significant improvements in postinfarct ventricular function in rodent models. Because reprogramming factors effective in transdifferentiating rodent cells are not sufficient to reprogram human cells, we sought to identify reprogramming factors potentially applicable to human studies. Lentivirus vectors expressing Gata4, Mef2c, and Tbx5 (GMT); Hand2 (H), Myocardin (My), or microRNA (miR)-590 were administered to rat, porcine, and human cardiac fibroblasts in vitro. induced cardiomyocyte-like cell production was then evaluated by assessing expression of the cardiomyocyte marker, cardiac troponin T (cTnT), whereas signaling pathway studies were performed to identify reprogramming factor targets. GMT administration induced cTnT expression in ≈6% of rat fibroblasts, but failed to induce cTnT expression in porcine or human cardiac fibroblasts. Addition of H/My and/or miR-590 to GMT administration resulted in cTNT expression in ≈5% of porcine and human fibroblasts and also upregulated the expression of the cardiac genes, MYH6 and TNNT2. When cocultured with murine cardiomyocytes, cTnT-expressing porcine cardiac fibroblasts exhibited spontaneous contractions. Administration of GMT plus either H/My or miR-590 alone also downregulated fibroblast genes COL1A1 and COL3A1. miR-590 was shown to directly suppress the zinc finger protein, specificity protein 1 (Sp1), which was able to substitute for miR-590 in inducing cellular reprogramming. These data support porcine studies as a surrogate for testing human cardiac reprogramming, and suggest that miR-590-mediated repression of Sp1 represents an alternative pathway for enhancing human cardiac cellular reprogramming. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Interleukin-35 administration counteracts established murine type 1 diabetes--possible involvement of regulatory T cells.

PubMed

Singh, Kailash; Kadesjö, Erik; Lindroos, Julia; Hjort, Marcus; Lundberg, Marcus; Espes, Daniel; Carlsson, Per-Ola; Sandler, Stellan; Thorvaldson, Lina

2015-07-30

The anti-inflammatory cytokine IL-35 is produced by regulatory T (Treg) cells to suppress autoimmune and inflammatory responses. The role of IL-35 in type 1 diabetes (T1D) remains to be answered. To elucidate this, we investigated the kinetics of Treg cell response in the multiple low dose streptozotocin induced (MLDSTZ) T1D model and measured the levels of IL-35 in human T1D patients. We found that Treg cells were increased in MLDSTZ mice. However, the Treg cells showed a decreased production of anti-inflammatory (IL-10, IL-35, TGF-β) and increased pro-inflammatory (IFN-γ, IL-2, IL-17) cytokines, indicating a phenotypic shift of Treg cells under T1D condition. IL-35 administration effectively both prevented development of, and counteracted established MLDSTZ T1D, seemingly by induction of Eos expression and IL-35 production in Treg cells, thus reversing the phenotypic shift of the Treg cells. IL-35 administration reversed established hyperglycemia in NOD mouse model of T1D. Moreover, circulating IL-35 levels were decreased in human T1D patients compared to healthy controls. These findings suggest that insufficient IL-35 levels play a pivotal role in the development of T1D and that treatment with IL-35 should be investigated in treatment of T1D and other autoimmune diseases.

Cell Permeability, Migration, and Reactive Oxygen Species Induced by Multi-Walled Carbon Nanotubes in Human Microvascular Endothelial Cells

PubMed Central

Pacurari, M; Qian, Y; Fu, W; Schwegler-Berry, D; Ding, M; Castranova, V; Guo, NL

2011-01-01

Multi-walled carbon nanotubes (MWCNT) have elicited great interest in biomedical applications due to their extraordinary physical, chemical, and optical properties. Intravenous administration of MWCNT-based medical imaging agents and drugs in animal models was utilized. However, the potential harmful health effects of MWCNT administration in humans have not yet been elucidated. Furthermore, to date, there are no apparent reports regarding the precise mechanisms of translocation of MWCNT into target tissues and organs from blood circulation. This study demonstrates that exposure to MWCNT leads to an increase in cell permeability in human microvascular endothelial cells (HMVEC). The results obtained from this study also showed that the MWCNT-induced rise in endothelial permeability is mediated by reactive oxygen species (ROS) production and actin filament remodeling. In addition, it was found that MWCNT promoted cell migration in HMVEC. Mechanistically, MWCNT exposure elevated the levels of monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule 1 (ICAM-1) in HMVEC. Taken together, these results provide new insights into the bioreactivity of MWCNT, which may have implications in the biomedical application of MWCNT in vascular targeting, imaging, and drug delivery. The results generated from this study also elucidate the potential adverse effects of MWCNT exposure on humans at the cellular level. PMID:22129238

21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

Code of Federal Regulations, 2011 CFR

2011-04-01

... chemically defined components (e.g., amino acids, vitamins, inorganic salts) that are essential for the ex... ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY...

21 CFR 1271.420 - HCT/Ps offered for import.

Code of Federal Regulations, 2010 CFR

2010-04-01

... recipient for reproductive use. (d) This section does not apply to peripheral blood stem/progenitor cells... peripheral blood stem/progenitor cells may present an unreasonable risk of communicable disease transmission...) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES...

21 CFR 1271.420 - HCT/Ps offered for import.

Code of Federal Regulations, 2011 CFR

2011-04-01

... recipient for reproductive use. (d) This section does not apply to peripheral blood stem/progenitor cells... peripheral blood stem/progenitor cells may present an unreasonable risk of communicable disease transmission...) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES...

21 CFR 1271.420 - HCT/Ps offered for import.

Code of Federal Regulations, 2014 CFR

2014-04-01

... recipient for reproductive use. (d) This section does not apply to peripheral blood stem/progenitor cells... peripheral blood stem/progenitor cells may present an unreasonable risk of communicable disease transmission...) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES...

21 CFR 1271.420 - HCT/Ps offered for import.

Code of Federal Regulations, 2013 CFR

2013-04-01

... recipient for reproductive use. (d) This section does not apply to peripheral blood stem/progenitor cells... peripheral blood stem/progenitor cells may present an unreasonable risk of communicable disease transmission...) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES...

21 CFR 1271.420 - HCT/Ps offered for import.

Code of Federal Regulations, 2012 CFR

2012-04-01

... recipient for reproductive use. (d) This section does not apply to peripheral blood stem/progenitor cells... peripheral blood stem/progenitor cells may present an unreasonable risk of communicable disease transmission...) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES...

Differentiation stage-specific regulation of primitive human hematopoietic progenitor cycling by exogenous and endogenous inhibitors in an in vivo model.

PubMed

Cashman, J D; Clark-Lewis, I; Eaves, A C; Eaves, C J

1999-12-01

Nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice transplanted with human cord blood or adult marrow cells and injected 6 weeks posttransplant with 2 daily doses of transforming growth factor-beta(1) (TGF-beta(1)), monocyte chemoattractant protein-1 (MCP-1), or a nonaggregating form of macrophage inflammatory protein-1alpha (MIP-1alpha) showed unique patterns of inhibition of human progenitor proliferation 1 day later. TGF-beta(1) was active on long-term culture initiating cells (LTC-IC) and on primitive erythroid and granulopoietic colony-forming cells (HPP-CFC), but had no effect on mature CFC. MCP-1 inhibited the cycling of both types of HPP-CFC but not LTC-IC. MIP-1alpha did not inhibit either LTC-IC or granulopoietic HPP-CFC but was active on erythroid HPP-CFC and mature granulopoietic CFC. All of these responses were independent of the source of human cells transplanted. LTC-IC of either human cord blood or adult marrow origin continue to proliferate in NOD/SCID mice for many weeks, although the turnover of all types of human CFC in mice transplanted with adult human marrow (but not cord blood) is downregulated after 6 weeks. Interestingly, administration of either MIP-1beta, an antagonist of both MIP-1alpha and MCP-1 or MCP-1(9-76), an antagonist of MCP-1 (and MCP-2 and MCP-3), into mice in which human marrow-derived CFC had become quiescent, caused the rapid reactivation of these progenitors in vivo. These results provide the first definition of stage-specific inhibitors of human hematopoietic progenitor cell cycling in vivo. In addition they show that endogenous chemokines can contribute to late graft failure, which can be reversed by the administration of specific antagonists.

In-vitro analysis of APA microcapsules for oral delivery of live bacterial cells.

PubMed

Chen, H; Ouyang, W; Jones, M; Haque, T; Lawuyi, B; Prakash, S

2005-08-01

Oral administration of microcapsules containing live bacterial cells has potential as an alternative therapy for several diseases. This article evaluates the suitability of the alginate-poly-L-lysine-alginate (APA) microcapsules for oral delivery of live bacterial cells, in-vitro, using a dynamic simulated human gastro-intestinal (GI) model. Results showed that the APA microcapsules were morphologically stable in the simulated stomach conditions, but did not retain their structural integrity after a 3-day exposure in simulated human GI media. The microbial populations of the tested bacterial cells and the activities of the tested enzymes in the simulated human GI suspension were not substantially altered by the presence of the APA microcapsules, suggesting that there were no significant adverse effects of oral administration of the APA microcapsules on the flora of the human gastrointestinal tract. When the APA microcapsules containing Lactobacillus plantarum 80 (LP80) were challenged in the simulated gastric medium (pH = 2.0), 80.0% of the encapsulated cells remained viable after a 5-min incubation; however, the viability decreased considerably (8.3%) after 15 min and dropped to 2.6% after 30 min and lower than 0.2% after 60 min, indicating the limitations of the currently obtainable APA membrane for oral delivery of live bacteria. Further in-vivo studies are required before conclusions can be made concerning the inadequacy of APA microcapsules for oral delivery of live bacterial cells.

AT13148, a first-in-class multi-AGC kinase inhibitor, potently inhibits gastric cancer cells both in vitro and in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xi, Yu; Department of General Surgery, First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi, Xinjiang 832008; Niu, Jianhua

The AGC kinase family is important cell proliferation and survival. Dysregulation of this family contributes to gastric cancer progression. Here, we evaluated the potential activity of AT13148, a first-in-class multi-AGC kinase inhibitor, against gastric cancer cells. Our results showed that AT13148 exerted potent cytotoxic and anti-proliferative activities against a panel human gastric cancer cell lines (HGC-27, AGS, SNU-601, N87 and MKN-28), possibly via inducing cancer cell apoptotic death. Apoptosis inhibition by the Caspase blockers dramatically attenuated AT13148-caused cytotoxicity against gastric cancer cells. Intriguingly, same AT13148 treatment was not cytotoxic/pro-apoptotic to the non-cancerous human gastric epithelial GEC-1 cells. At the signaling level,more » AT13148 treatment in gastric cancer cells dramatically suppressed activation of multiple AGC kinases, including Akt (at p-Thr-308), p70S6 kinase (p70S6K), glycogen synthase kinase 3β (GSK-3β) and p90 ribosomal S6 kinase (RSK). Our in vivo studies demonstrated that daily oral gavage of AT13148 at well-tolerated doses significantly inhibited HGC27 xenograft tumor growth in nude mice. AGC activity was also dramatically decreased in AT13148-administrated HGC27 tumors. Therefore, targeting AGC kinases by AT13148 demonstrates superior anti-gastric cancer activity both in vitro and in vivo. The preclinical results of this study support the progression of this molecule into future evaluation as a valuable anti-gastric cancer candidate. - Highlights: • AT13148 is cytotoxic and anti-proliferative to human gastric cancer cells. • AT13148 induces gastric cancer cell apoptotic death, inhibited by Caspase inhibitors. • AT13148 inactivates multiple AGC kinases in human gastric cancer cells. • AT13148 oral administration suppresses HGC27 xenograft growth in nude mice. • AT13148 oral administration inhibits multiple AGC kinases in HGC27 xenograft tumors.« less

The anti-obesity drug orlistat reveals anti-viral activity.

PubMed

Ammer, Elisabeth; Nietzsche, Sandor; Rien, Christian; Kühnl, Alexander; Mader, Theresa; Heller, Regine; Sauerbrei, Andreas; Henke, Andreas

2015-12-01

The administration of drugs to inhibit metabolic pathways not only reduces the risk of obesity-induced diseases in humans but may also hamper the replication of different viral pathogens. In order to investigate the value of the US Food and Drug Administration-approved anti-obesity drug orlistat in view of its anti-viral activity against different human-pathogenic viruses, several anti-viral studies, electron microscopy analyses as well as fatty acid uptake experiments were performed. The results indicate that administrations of non-cytotoxic concentrations of orlistat reduced the replication of coxsackievirus B3 (CVB3) in different cell types significantly. Moreover, orlistat revealed cell protective effects and modified the formation of multi-layered structures in CVB3-infected cells, which are necessary for viral replication. Lowering fatty acid uptake from the extracellular environment by phloretin administrations had only marginal impact on CVB3 replication. Finally, orlistat reduced also the replication of varicella-zoster virus moderately but had no significant influence on the replication of influenza A viruses. The data support further experiments into the value of orlistat as an inhibitor of the fatty acid synthase to develop new anti-viral compounds, which are based on the modulation of cellular metabolic pathways.

A fully human anti-Ep-CAM scFv-beta-glucuronidase fusion protein for selective chemotherapy with a glucuronide prodrug.

PubMed

de Graaf, M; Boven, E; Oosterhoff, D; van der Meulen-Muileman, I H; Huls, G A; Gerritsen, W R; Haisma, H J; Pinedo, H M

2002-03-04

Monoclonal antibodies against tumour-associated antigens could be useful to deliver enzymes selectively to the site of a tumour for activation of a non-toxic prodrug. A completely human fusion protein may be advantageous for repeated administration, as host immune responses may be avoided. We have constructed a fusion protein consisting of a human single chain Fv antibody, C28, against the epithelial cell adhesion molecule and the human enzyme beta-glucuronidase. The sequences encoding C28 and human enzyme beta-glucuronidase were joined by a sequence encoding a flexible linker, and were preceded by the IgGkappa signal sequence for secretion of the fusion protein. A CHO cell line was engineered to secrete C28-beta-glucuronidase fusion protein. Antibody specificity and enzyme activity were retained in the secreted fusion protein that had an apparent molecular mass of 100 kDa under denaturing conditions. The fusion protein was able to convert a non-toxic prodrug of doxorubicin, N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate to doxorubicin, resulting in cytotoxicity. A bystander effect was demonstrated, as doxorubicin was detected in all cells after N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate administration when only 10% of the cells expressed the fusion protein. This is the first fully human and functional fusion protein consisting of an scFv against epithelial cell adhesion molecule and human enzyme beta-glucuronidase for future use in tumour-specific activation of a non-toxic glucuronide prodrug. Copyright 2002 Cancer Research UK

Potential therapeutic effect of the secretome from human uterine cervical stem cells against both cancer and stromal cells compared with adipose tissue stem cells

PubMed Central

Seoane, Samuel; Bermúdez, María A.; Lamelas, Maria Luz; Garcia-Caballero, Tomás; Schneider, José; Perez-Fernandez, Roman; Vizoso, Francisco J.

2014-01-01

Evidences indicate that tumor development and progression towards a malignant phenotype depend not only on cancer cells themselves, but are also deeply influenced by tumor stroma reactivity. The present study uses mesenchymal stem cells from normal human uterine cervix (hUCESCs), isolated by the minimally invasive method of routine Pap cervical smear, to study their effect on the three main cell types in a tumor: cancer cells, fibroblasts and macrophages. Administration of hUCESCs-conditioned medium (CM) to a highly invasive breast cancer MDA-MB-231 cell line and to human breast tumors with high cell proliferation rates had the effect of reducing cell proliferation, modifying the cell cycle, inducing apoptosis, and decreasing invasion. In a xenograft mouse tumor model, hUCESCs-CM reduced tumor growth and increased overall survival. In cancer-associated fibroblasts, administration of hUCESCs-CM resulted in reduced cell proliferation, greater apoptosis and decreased invasion. In addition, hUCESCs-CM inhibited and reverted macrophage differentiation. The analysis of hUCESCs-CM (fresh and lyophilized) suggests that a complex paracrine signaling network could be implicated in the anti-tumor potential of hUCESCs. In light of their anti-tumor potential, the easy cell isolation method, and the fact that lyophilization of their CM conserves original properties make hUCESCs good candidates for experimental or clinical applications in anticancer therapy. PMID:25296979

21 CFR 1271.75 - How do I screen a donor?

Code of Federal Regulations, 2010 CFR

2010-04-01

...) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES..., you must screen a donor of cells or tissue by reviewing the donor's relevant medical records for: (1...) Communicable disease risks associated with xenotransplantation. (b) Donors of viable, leukocyte-rich cells or...

21 CFR 1271.75 - How do I screen a donor?

Code of Federal Regulations, 2014 CFR

2014-04-01

...) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES..., you must screen a donor of cells or tissue by reviewing the donor's relevant medical records for: (1...) Communicable disease risks associated with xenotransplantation. (b) Donors of viable, leukocyte-rich cells or...

In vitro and in vivo studies on antitumor effects of gossypol on human stomach adenocarcinoma (AGS) cell line and MNNG induced experimental gastric cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gunassekaran, G.R., E-mail: gunassekaran@yahoo.co.in; Kalpana Deepa Priya, D.; Gayathri, R.

2011-08-12

Highlights: {yields} Gossypol is a well known polyphenolic compound used for anticancer studies but we are the first to report that gossypol has antitumor effect on MNNG induced gastric cancer in experimental animal models. {yields} Our study shows that gossypol inhibits the proliferation of AGS (human gastric adenocarcinoma) cell line. {yields} In animal models, gossypol extends the survival of cancer bearing animals and also protects the cells from carcinogenic effect. {yields} So we suggest that gossypol would be a potential chemotherapeutic and chemopreventive agent for gastric cancer. -- Abstract: The present study has evaluated the chemopreventive effects of gossypol onmore » N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinogenesis and on human gastric adenocarcinoma (AGS) cell line. Gossypol, C{sub 30}H{sub 30}O{sub 8}, is a polyphenolic compound that has anti proliferative effect and induces apoptosis in various cancer cells. The aim of this work was to delineate in vivo and in vitro anti-initiating mechanisms of orally administered gossypol in target (stomach) tissues and in human gastric adenocarcinoma (AGS) cell line. In vitro results prove that gossypol has potent cytotoxic effect and inhibit the proliferation of adenocarcinoma (AGS) cell line. In vivo results prove gossypol to be successful in prolonging the survival of MNNG induced cancer bearing animals and in delaying the onset of tumor in animals administrated with gossypol and MNNG simultaneously. Examination of the target (stomach) tissues in sacrificed experimental animals shows that administration of gossypol significantly reduces the level of tumor marker enzyme (carcino embryonic antigen) and pepsin. The level of Nucleic acid contents (DNA and RNA) significantly reduces, and the membrane damage of glycoprotein subsides, in the target tissues of cancer bearing animals, with the administration of gossypol. These data suggest that gossypol may create a beneficial effect in patients with gastric cancer.« less

Differential effects of oestrogenic hormones on cell proliferation in the colonic crypt epithelium and in colonic carcinomata of rats.

PubMed

Tutton, P J; Barkla, D H

1982-01-01

A number of hormones, including some steroids, have previously been shown to influence the rate of cell division in the colonic crypt epithelium and in colonic tumours. In this report the effect of oophorectomy and of treatment with ovarian hormones on cell proliferation in these tissues is compared. Colonic tumours cell proliferation was retarded following oophorectomy and this retardation was reversed by the administration of oestradiol, but not by the administration of progesterone. Oophorectomy did not retard cell proliferation in the colonic crypts. The possible significance of these findings in relation to age-dependent variations in the sex ratio for human bowel cancer is discussed.

Distribution of subpopulations of dendritic cells in peripheral blood of patients treated with exogenous thyrotropin

PubMed Central

2012-01-01

Background Dendritic cells (DCs) play a major role as regulators of inflammatory events associated with thyroid pathology. The immunoregulatory function of DCs depends strongly on their subtype, as well as maturation and activation status. Numerous hormonal factors modulate the immune properties of DCs, however, little is known about effects exerted by the hypothalamus-pituitary-thyroid-axis. Recently, we have shown a direct regulatory influence of thyroid hormones (TH) on human DCs function. The aim of the present study was to analyze the effect of systemically administered thyrotropin (TSH) on human blood DCs ex vivo. Methods Blood samples for the cytometric analysis of peripheral blood plasmacytoid and myeloid DCs subtypes were collected from patients subjected to total thyroidectomy because of differentiated thyroid carcinoma at 2 time points: (i) directly before the commencement of TSH administration and (ii) 5 days after first TSH injection. The whole blood quantitative and phenotypic analysis of plasmacytoid and myeloid DCs subtypes was performed by flow cytometry. Results Administration of TSH did not influence the percentage of plasmacytoid DCs in peripheral blood of study participants. Also the percentage of the two main myeloid DCs subpopulations – CD1c/BDCA1+ DCs and CD141/BDCA3+ DCs did not change significantly. TSH administration had no effect on the surface expression of CD86 – one of the major costimulatory molecules – neither in the whole peripheral blood mononuclear cell (PBMC) fraction nor in particular DCs subtypes. Conclusions In the present study, we demonstrated no influence of systemic TSH administration on human peripheral blood DCs subtypes. These results are in accordance with our previous work suggesting the direct effect of TH on human DCs ex vivo. PMID:23199104

Distribution of subpopulations of dendritic cells in peripheral blood of patients treated with exogenous thyrotropin.

PubMed

Stasiołek, Mariusz; Adamczewski, Zbigniew; Puła, Bartosz; Krawczyk-Rusiecka, Kinga; Zygmunt, Arkadiusz; Borowiecka, Magdalena; Dzięgiel, Piotr; Lewiński, Andrzej

2012-11-30

Dendritic cells (DCs) play a major role as regulators of inflammatory events associated with thyroid pathology. The immunoregulatory function of DCs depends strongly on their subtype, as well as maturation and activation status. Numerous hormonal factors modulate the immune properties of DCs, however, little is known about effects exerted by the hypothalamus-pituitary-thyroid-axis. Recently, we have shown a direct regulatory influence of thyroid hormones (TH) on human DCs function. The aim of the present study was to analyze the effect of systemically administered thyrotropin (TSH) on human blood DCs ex vivo. Blood samples for the cytometric analysis of peripheral blood plasmacytoid and myeloid DCs subtypes were collected from patients subjected to total thyroidectomy because of differentiated thyroid carcinoma at 2 time points: (i) directly before the commencement of TSH administration and (ii) 5 days after first TSH injection. The whole blood quantitative and phenotypic analysis of plasmacytoid and myeloid DCs subtypes was performed by flow cytometry. Administration of TSH did not influence the percentage of plasmacytoid DCs in peripheral blood of study participants. Also the percentage of the two main myeloid DCs subpopulations - CD1c/BDCA1+ DCs and CD141/BDCA3+ DCs did not change significantly. TSH administration had no effect on the surface expression of CD86 - one of the major costimulatory molecules - neither in the whole peripheral blood mononuclear cell (PBMC) fraction nor in particular DCs subtypes. In the present study, we demonstrated no influence of systemic TSH administration on human peripheral blood DCs subtypes. These results are in accordance with our previous work suggesting the direct effect of TH on human DCs ex vivo.

21 CFR 1271.15 - Are there any exceptions from the requirements of this part?

Code of Federal Regulations, 2010 CFR

2010-04-01

... ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS General Provisions § 1271.15 Are... only recovers reproductive cells or tissue and immediately transfers them into a sexually intimate partner of the cell or tissue donor. (f) You are not required to register or list your HCT/P's...

21 CFR 1271.15 - Are there any exceptions from the requirements of this part?

Code of Federal Regulations, 2011 CFR

2011-04-01

... ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS General Provisions § 1271.15 Are... only recovers reproductive cells or tissue and immediately transfers them into a sexually intimate partner of the cell or tissue donor. (f) You are not required to register or list your HCT/P's...

21 CFR 1271.15 - Are there any exceptions from the requirements of this part?

Code of Federal Regulations, 2014 CFR

2014-04-01

... ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS General Provisions § 1271.15 Are... only recovers reproductive cells or tissue and immediately transfers them into a sexually intimate partner of the cell or tissue donor. (f) You are not required to register or list your HCT/P's...

Plant-based oral delivery of β-glucocerebrosidase as an enzyme replacement therapy for Gaucher's disease.

PubMed

Shaaltiel, Yoseph; Gingis-Velitski, Svetlana; Tzaban, Salit; Fiks, Nadia; Tekoah, Yoram; Aviezer, David

2015-10-01

Gaucher's disease (GD), a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy (ERT) using recombinant GCD that is administered intravenously every 2 weeks. However, intravenous administration includes discomfort or pain and might cause local and systemic infections that may lead to low patient compliance. An orally administered drug has the potential to alleviate these problems. In this study, we describe the potential use of plant cells as a vehicle for the oral delivery of recombinant human GCD (prGCD) expressed in carrot cells. The in vitro results demonstrate that the plant cells protect the recombinant protein in the gastric fluids and may enable absorption into the blood. Feeding experiments, with rat and pig as model animals, using carrot cells containing prGCD, show that active recombinant prGCD was found in the digestive tract and blood system and reached both, liver and spleen, the target organs in GD. These results demonstrate that the oral administration of proteins encapsulated in plant cells is feasible. Specifically, carrot cells containing recombinant human prGCD can be used as an oral delivery system and are a feasible alternative to intravenous administration of ERT for GD. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

A fully human anti-Ep-CAM scFv-beta-glucuronidase fusion protein for selective chemotherapy with a glucuronide prodrug

PubMed Central

de Graaf, M; Boven, E; Oosterhoff, D; van der Meulen-Muileman, I H; Huls, G A; Gerritsen, W R; Haisma, H J; Pinedo, H M

2002-01-01

Monoclonal antibodies against tumour-associated antigens could be useful to deliver enzymes selectively to the site of a tumour for activation of a non-toxic prodrug. A completely human fusion protein may be advantageous for repeated administration, as host immune responses may be avoided. We have constructed a fusion protein consisting of a human single chain Fv antibody, C28, against the epithelial cell adhesion molecule and the human enzyme β-glucuronidase. The sequences encoding C28 and human enzyme β-glucuronidase were joined by a sequence encoding a flexible linker, and were preceded by the IgGκ signal sequence for secretion of the fusion protein. A CHO cell line was engineered to secrete C28-β-glucuronidase fusion protein. Antibody specificity and enzyme activity were retained in the secreted fusion protein that had an apparent molecular mass of 100 kDa under denaturing conditions. The fusion protein was able to convert a non-toxic prodrug of doxorubicin, N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-β-glucuronyl carbamate to doxorubicin, resulting in cytotoxicity. A bystander effect was demonstrated, as doxorubicin was detected in all cells after N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-β-glucuronyl carbamate administration when only 10% of the cells expressed the fusion protein. This is the first fully human and functional fusion protein consisting of an scFv against epithelial cell adhesion molecule and human enzyme β-glucuronidase for future use in tumour-specific activation of a non-toxic glucuronide prodrug. British Journal of Cancer (2002) 86, 811–818. DOI: 10.1038/sj/bjc/6600143 www.bjcancer.com © 2002 Cancer Research UK PMID:11875747

A potential oral anticancer drug candidate, Moringa oleifera leaf extract, induces the apoptosis of human hepatocellular carcinoma cells

PubMed Central

JUNG, IL LAE; LEE, JU HYE; KANG, SE CHAN

2015-01-01

It has previously been reported that cold water-extracts of Moringa oleifera leaf have anticancer activity against various human cancer cell lines, including non-small cell lung cancer. In the present study, the anticancer activity of M. oleifera leaf extracts was investigated in human hepatocellular carcinoma HepG2 cells. By the analysis of apoptotic signals, including the induction of caspase or poly(ADP-ribose) polymerase cleavage, and the Annexin V and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays, it was demonstrated that M. oleifera leaf extracts induce the apoptosis of HepG2 cells. In the hollow fiber assay, oral administration of the leaf extracts significantly reduced (44–52%) the proliferation of the HepG2 cells and A549 non-small cell lung cancer cells. These results support the potential of soluble extracts of M. oleifera leaf as orally administered therapeutics for the treatment of human liver and lung cancers. PMID:26622717

Blockade of epidermal growth factor receptor signaling in tumor cells and tumor-associated endothelial cells for therapy of androgen-independent human prostate cancer growing in the bone of nude mice.

PubMed

Kim, Sun-Jin; Uehara, Hisanori; Karashima, Takashi; Shepherd, David L; Killion, Jerald J; Fidler, Isaiah J

2003-03-01

We determined whether blockade of the epidermal growth factor receptor (EGF-R) signaling pathway by oral administration of the EGF-R tyrosine kinase inhibitor (PKI 166) alone or in combination with injectable Taxol inhibits the growth of PC-3MM2 human prostate cancer cells in the bone of nude mice. Male nude mice implanted with PC-3MM2 cells in the tibia were treated with oral administrations of PKI 166 or PKI 166 plus injectable Taxol beginning 3 days after implantation. The incidence and size of bone tumors and destruction of bone were determined by digitalized radiography. Expression of epidermal growth factor (EGF), EGF-R, and activated EGF-R in tumor cells and tumor-associated endothelial cells was determined by immunohistochemistry. Oral administration of PKI 166 or PKI 166 plus injectable Taxol reduced the incidence and size of bone tumors and destruction of bone. Immunohistochemical analysis revealed that PC-3MM2 cells growing adjacent to the bone expressed high levels of EGF and activated EGF-R, whereas tumor cells in the adjacent musculature did not. Moreover, endothelial cells within the bone tumor lesions, but not in uninvolved bone or tumors in the muscle, expressed high levels of activated EGF-R. Treatment with PKI 166 and more so with PKI 166 plus Taxol significantly inhibited phosphorylation of EGF-R on tumor and endothelial cells and induced significant apoptosis and endothelial cells within tumor lesions. These data indicate that endothelial cells exposed to EGF produced by tumor cells express activated EGF-R and that targeting EGF-R can produce significant therapeutic effects against prostate cancer bone metastasis.

Cultured networks of excitatory projection neurons and inhibitory interneurons for studying human cortical neurotoxicity

PubMed Central

Xu, Jin-Chong; Fan, Jing; Wang, Xueqing; Eacker, Stephen M.; Kam, Tae-In; Chen, Li; Yin, Xiling; Zhu, Juehua; Chi, Zhikai; Jiang, Haisong; Chen, Rong; Dawson, Ted M.; Dawson, Valina L.

2017-01-01

Translating neuroprotective treatments from discovery in cell and animal models to the clinic has proven challenging. To reduce the gap between basic studies of neurotoxicity and neuroprotection and clinically relevant therapies, we developed a human cortical neuron culture system from human embryonic stem cells (ESCs) or inducible pluripotent stem cells (iPSCs) that generated both excitatory and inhibitory neuronal networks resembling the composition of the human cortex. This methodology used timed administration of retinoic acid (RA) to FOXG1 neural precursor cells leading to differentiation of neuronal populations representative of the six cortical layers with both excitatory and inhibitory neuronal networks that were functional and homeostatically stable. In human cortical neuron cultures, excitotoxicity or ischemia due to oxygen and glucose deprivation led to cell death that was dependent on N-methyl-D-aspartate (NMDA) receptors, nitric oxide (NO), and the poly (ADP-ribose) polymerase (PARP)-dependent cell death, a cell death pathway designated parthanatos to separate it from apoptosis, necroptosis and other forms of cell death. Neuronal cell death was attenuated by PARP inhibitors that are currently in clinical trials for cancer treatment. This culture system provides a new platform for the study of human cortical neurotoxicity and suggests that PARP inhibitors may be useful for ameliorating excitotoxic and ischemic cell death in human neurons. PMID:27053772

Telomerase reverse transcriptase (TERT) expression and role of vincristine sulfate in mouse model of malignancy related peritoneal ascites: an experimental metastatic condition.

PubMed

Chaklader, M; Das, P; Pereira, J A; Chatterjee, S; Basak, P; Law, A; Banerjee, T; Chauhan, S; Law, S

2011-06-01

To evaluate the efficacy of intraperitoneal vincristine administration into ascitic sarcoma-180 bearing mice as a model of human malignant ascites regarding various peritoneal/retroperitoneal sarcomatosis, and to evaluate the flowcytometric telomerase reverse transcriptase expression for the diagnostic and prognostic purposes. Present study included disease induction by intraperitoneal homologous ascitic sarcoma-180 transplantation followed by in vivo intraperitoneal drug administration to study mitotic index, flowcytometric cell cycle and telomerase reverse transcriptase expression pattern, erythrosin-B dye exclusion study for malignant cell viability assessment. Besides, in vitro malignant ascite culture in presence and absence of vincristine sulfate and survival study were also taken into consideration. Intraperitoneal vincristine administration (concentration 0.5 mg/kg body weight) significantly diminished the mitotic index in diseased subjects in comparison to untreated control subjects. Treated group of animals showed increased life span and median survival time. Cell viability assessment during the course of drug administration also revealed gradual depression on cell viability over time. Flowcytometric cell cycle analysis showed a good prognostic feature of chemotherapeutic administration schedule by representing high G2/M phase blocked cells along with reduced telomerase reverse transcriptase positive cells in treated animals. We conclude that long term administration of vincristine sulfate in small doses could be a good pharmacological intervention in case of malignant peritoneal ascites due to sarcomatosis as it indirectly reduced the level of telomerase reverse transcriptase expression in malignant cells by directly regulating cell cycle and simultaneously increased the life expectancy of the diseased subjects.

BMP7 Induces Dormancy of Prostatic Tumor Stem Cell in Bone

DTIC Science & Technology

2012-10-01

the upper back of nude mice. Recombinant human BMP7 was peritumorally injected daily after implantation. Tumor growth was monitored weekly by...of BMP7 was administrated daily through i.v. after intracardiac injection of CSCs from PC3mm or C4-2B cells to the mice. As shown in Figure 5A...mice, and then BMP7 was administrated daily . BLI of representative mice in each group six weeks after implantation (A). Normalized BLI signals

Induction of proteinases in the human preovulatory follicle of the menstrual cycle by human chorionic gonadotropin.

PubMed

Rosewell, Katherine L; Al-Alem, Linah; Zakerkish, Farnosh; McCord, Lauren; Akin, James W; Chaffin, Charles L; Brännström, Mats; Curry, Thomas E

2015-03-01

To explore the temporal expression in granulosa and theca cells of key members of the MMP and ADAMTS families across the periovulatory period in women to gain insight into their possible roles during ovulation and early luteinization. Experimental prospective clinical study and laboratory-based investigation. University medical center and private IVF center. Thirty-eight premenopausal women undergoing surgery for tubal ligation and six premenopausal women undergoing assisted reproductive techniques. Administration of hCG and harvesting of follicles by laparoscopy and collection of granulosa-lutein cells at oocyte retrieval. Expression of mRNA for matrix metalloproteinase (MMPs) and the A disintegrin and metalloproteinase with thrombospondin-like motifs (ADAMTS) in human granulosa cells and theca cells collected across the periovulatory period of the menstrual cycle and in cultured granulosa-lutein cells after hCG. Localization of MMPs and ADAMTSs by immunohistochemistry. Expression of MMP1 and MMP19 mRNA increased in both granulosa and theca cells after hCG administration. ADAMTS1 and ADAMTS9 mRNA increased in granulosa cells after hCG treatment, however, thecal cell expression for ADAMTS1 was unchanged, while ADAMTS9 expression was decreased. Expression of MMP8 and MMP13 mRNA was unchanged. Immunohistochemistry confirmed the localization of MMP1, MMP19, ADAMTS1, and ADAMTS9 to the granulosa and thecal cell layers. The collection of the dominant follicle throughout the periovulatory period has allowed the identification of proteolytic remodeling enzymes in the granulosa and theca compartments that may be critically involved in human ovulation. These proteinases may work in concert to regulate breakdown of the follicular wall and release of the oocyte. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

21 CFR 1271.440 - Orders of retention, recall, destruction, and cessation of manufacturing.

Code of Federal Regulations, 2010 CFR

2010-04-01

... FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS Inspection... protections against risks of communicable disease transmission; or the HCT/P is infected or contaminated so as... communicable disease transmission, the Food and Drug Administration (FDA) may take one or more of the following...

Immunotherapy of BALB/c mice bearing Ehrlich ascites tumor with vitamin D-binding protein-derived macrophage activating factor.

PubMed

Yamamoto, N; Naraparaju, V R

1997-06-01

Vitamin D3-binding protein (DBP; human DBP is known as Gc protein) is the precursor of macrophage activating factor (MAF). Treatment of mouse DBP with immobilized beta-galactosidase or treatment of human Gc protein with immobilized beta-galactosidase and sialidase generated a remarkably potent MAF, termed DBPMAF or GcMAF, respectively. The domain of Gc protein responsible for macrophage activation was cloned and enzymatically converted to the cloned MAF, designated CdMAF. In Ehrlich ascites tumor-bearing mice, tumor-specific serum alpha-N-acetylgalactosaminidase (NaGalase) activity increased linearly with time as the transplanted tumor cells grew in the peritoneal cavity. Therapeutic effects of DBPMAF, GcMAF, and CdMAF on mice bearing Ehrlich ascites tumor were assessed by survival time, the total tumor cell count in the peritoneal cavity, and serum NaGalase activity. Mice that received a single administration of DBPMAF or GcMAF (100 pg/mouse) on the same day after transplantation of tumor (1 x 10(5) cells) showed a mean survival time of 35 +/- 4 days, whereas tumor-bearing controls had a mean survival time of 16 +/- 2 days. When mice received the second DBPMAF or GcMAF administration at day 4, they survived more than 50 days. Mice that received two DBPMAF administrations, at days 4 and 8 after transplantation of 1 x 10(5) tumor cells, survived up to 32 +/- 4 days. At day 4 posttransplantation, the total tumor cell count in the peritoneal cavity was approximately 5 x 10(5) cells. Mice that received two DBPMAF administrations, at days 0 and 4 after transplantation of 5 x 10(5) tumor cells, also survived up to 32 +/- 4 days, while control mice that received the 5 x 10(5) ascites tumor cells only survived for 14 +/- 2 days. Four DBPMAF, GcMAF, or CdMAF administrations to mice transplanted with 5 x 10(5) Ehrlich ascites tumor cells with 4-day intervals showed an extended survival of at least 90 days and an insignificantly low serum NaGalase level between days 30 and 90.

Activity of a new nitrosourea (TCNU) in human lung cancer xenografts.

PubMed Central

Fergusson, R. J.; Anderson, L. E.; Macpherson, J. S.; Robins, P.; Smyth, J. F.

1988-01-01

The activity of a new nitrosourea (TCNU) based on the endogenous amino acid taurine was assessed in three human lung cancer xenografts growing in immunodeficient mice. Moderate activity (specific growth delays of 0.63 and 1.13 compared with controls) was seen in two non-small cell tumours after a single oral administration of 20 mg-1kg. This dose was curative in a small cell xenograft. By using high performance liquid chromatography it was possible to detect parent drug in the tumours as well as the plasma and tissues after oral administration of TCNU. Drug sensitivity was correlated inversely with the amount of the DNA repair enzyme 0(6)-methylguanine-DNA methyltransferase assayed from extracts of the tumour cells but not with the levels of parent drug within the tumour. This compound appears to have unique pharmacokinetic properties compared with other chloroethylnitrosoureas. PMID:3390369

Preclinical safety testing for cell-based products using animals.

PubMed

McBlane, James W

2015-09-01

The objectives of preclinical testing include to show why there might be therapeutic benefit in patients and to provide information on the product's toxicity. For cell-based products, given even once, there may be long term exposure and this could imply, unlike for conventional drugs, that all preclinical studies may be needed prior to first human use. The duration of exposure to cells should be studied in animals to guide toxicity assessments. Distribution of cells after administration by a route resembling that intended in humans should be studied to understand potential risks. Risk of tumour formation with the product may also need to be characterised. To the extent that this information can be generated by in vitro testing, studies in animals may not be needed and limitations on the capability of preclinical data to predict human toxicity are recognised: species-specificity make some cell products act only in humans and a human cell-product might be expected to be rejected by immunocompetent animals. Does this suggest testing in immunosuppressed animals or of development of an animal-cell product supposedly similar to the human cell product? No single answer seems to fit every situation. Copyright © 2015.

I.V. infusion of brain-derived neurotrophic factor gene-modified human mesenchymal stem cells protects against injury in a cerebral ischemia model in adult rat.

PubMed

Nomura, T; Honmou, O; Harada, K; Houkin, K; Hamada, H; Kocsis, J D

2005-01-01

I.V. delivery of mesenchymal stem cells prepared from adult bone marrow reduces infarction size and ameliorates functional deficits in rat cerebral ischemia models. Administration of the brain-derived neurotrophic factor to the infarction site has also been demonstrated to be neuroprotective. To test the hypothesis that brain-derived neurotrophic factor contributes to the therapeutic benefits of mesenchymal stem cell delivery, we compared the efficacy of systemic delivery of human mesenchymal stem cells and human mesenchymal stem cells transfected with a fiber-mutant F/RGD adenovirus vector with a brain-derived neurotrophic factor gene (brain-derived neurotrophic factor-human mesenchymal stem cells). A permanent middle cerebral artery occlusion was induced by intraluminal vascular occlusion with a microfilament. Human mesenchymal stem cells and brain-derived neurotrophic factor-human mesenchymal stem cells were i.v. injected into the rats 6 h after middle cerebral artery occlusion. Lesion size was assessed at 6 h, 1, 3 and 7 days using MR imaging, and histological methods. Functional outcome was assessed using the treadmill stress test. Both human mesenchymal stem cells and brain-derived neurotrophic factor-human mesenchymal stem cells reduced lesion volume and elicited functional improvement compared with the control sham group, but the effect was greater in the brain-derived neurotrophic factor-human mesenchymal stem cell group. ELISA analysis of the infarcted hemisphere revealed an increase in brain-derived neurotrophic factor in the human mesenchymal stem cell groups, but a greater increase in the brain-derived neurotrophic factor-human mesenchymal stem cell group. These data support the hypothesis that brain-derived neurotrophic factor contributes to neuroprotection in cerebral ischemia and cellular delivery of brain-derived neurotrophic factor can be achieved by i.v. delivery of human mesenchymal stem cells.

Fluorescent nanodiamonds enable quantitative tracking of human mesenchymal stem cells in miniature pigs

NASA Astrophysics Data System (ADS)

Su, Long-Jyun; Wu, Meng-Shiue; Hui, Yuen Yung; Chang, Be-Ming; Pan, Lei; Hsu, Pei-Chen; Chen, Yit-Tsong; Ho, Hong-Nerng; Huang, Yen-Hua; Ling, Thai-Yen; Hsu, Hsao-Hsun; Chang, Huan-Cheng

2017-03-01

Cell therapy is a promising strategy for the treatment of human diseases. While the first use of cells for therapeutic purposes can be traced to the 19th century, there has been a lack of general and reliable methods to study the biodistribution and associated pharmacokinetics of transplanted cells in various animal models for preclinical evaluation. Here, we present a new platform using albumin-conjugated fluorescent nanodiamonds (FNDs) as biocompatible and photostable labels for quantitative tracking of human placenta choriodecidual membrane-derived mesenchymal stem cells (pcMSCs) in miniature pigs by magnetic modulation. With this background-free detection technique and time-gated fluorescence imaging, we have been able to precisely determine the numbers as well as positions of the transplanted FND-labeled pcMSCs in organs and tissues of the miniature pigs after intravenous administration. The method is applicable to single-cell imaging and quantitative tracking of human stem/progenitor cells in rodents and other animal models as well.

21 CFR 1271.80 - What are the general requirements for donor testing?

Code of Federal Regulations, 2010 CFR

2010-04-01

... ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS Donor Eligibility § 1271.80 What are... donor specimen for testing at the time of recovery of cells or tissue from the donor; or up to 7 days before or after recovery, except: (1) For donors of peripheral blood stem/progenitor cells, bone marrow...

21 CFR 1271.80 - What are the general requirements for donor testing?

Code of Federal Regulations, 2012 CFR

2012-04-01

... donor specimen for testing at the time of recovery of cells or tissue from the donor; or up to 7 days before or after recovery, except: (1) For donors of peripheral blood stem/progenitor cells, bone marrow... ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS Donor Eligibility § 1271.80 What are...

21 CFR 1271.80 - What are the general requirements for donor testing?

Code of Federal Regulations, 2013 CFR

2013-04-01

... donor specimen for testing at the time of recovery of cells or tissue from the donor; or up to 7 days before or after recovery, except: (1) For donors of peripheral blood stem/progenitor cells, bone marrow... ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS Donor Eligibility § 1271.80 What are...

21 CFR 1271.80 - What are the general requirements for donor testing?

Code of Federal Regulations, 2014 CFR

2014-04-01

... donor specimen for testing at the time of recovery of cells or tissue from the donor; or up to 7 days before or after recovery, except: (1) For donors of peripheral blood stem/progenitor cells, bone marrow... ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS Donor Eligibility § 1271.80 What are...

21 CFR 1271.80 - What are the general requirements for donor testing?

Code of Federal Regulations, 2011 CFR

2011-04-01

... donor specimen for testing at the time of recovery of cells or tissue from the donor; or up to 7 days before or after recovery, except: (1) For donors of peripheral blood stem/progenitor cells, bone marrow... ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS Donor Eligibility § 1271.80 What are...

An inducible caspase 9 safety switch for T-cell therapy

PubMed Central

Straathof, Karin C.; Pulè, Martin A.; Yotnda, Patricia; Dotti, Gianpietro; Vanin, Elio F.; Brenner, Malcolm K.; Heslop, Helen E.; Spencer, David M.; Rooney, Cliona M.

2005-01-01

The efficacy of adoptive T-cell therapy as treatment for malignancies may be enhanced by genetic modification of infused cells. However, oncogenic events due to vector/transgene integration, and toxicities due to the infused cells themselves, have tempered enthusiasm. A safe and efficient means of removing aberrant cells in vivo would ameliorate these concerns. We describe a “safety switch” that can be stably and efficiently expressed in human T cells without impairing phenotype, function, or antigen specificity. This reagent is based on a modified human caspase 9 fused to a human FK506 binding protein (FKBP) to allow conditional dimerization using a small molecule pharmaceutical. A single 10-nM dose of synthetic dimerizer drug induces apoptosis in 99% of transduced cells selected for high transgene expression in vitro and in vivo. This system has several advantages over currently available suicide genes. First, it consists of human gene products with low potential immunogenicity. Second, administration of dimerizer drug has no effects other than the selective elimination of transduced T cells. Third, inducible caspase 9 maintains function in T cells overexpressing antiapoptotic molecules. These characteristics favor incorporation of inducible caspase 9 as a safety feature in human T-cell therapies. PMID:15728125

The clinical use of regenerative therapy in COPD

PubMed Central

Lipsi, Roberto; Rogliani, Paola; Calzetta, Luigino; Segreti, Andrea; Cazzola, Mario

2014-01-01

Regenerative or stem cell therapy is an emerging field of treatment based on stimulation of endogenous resident stem cells or administration of exogenous stem cells to treat diseases or injury and to replace malfunctioning or damaged tissues. Current evidence suggests that in the lung, these cells may participate in tissue homeostasis and regeneration after injury. Animal and human studies have demonstrated that tissue-specific stem cells and bone marrow-derived cells contribute to lung tissue regeneration and protection, and thus administration of exogenous stem/progenitor cells or humoral factors responsible for the activation of endogenous stem/progenitor cells may be a potent next-generation therapy for chronic obstructive pulmonary disease. The use of bone marrow-derived stem cells could allow repairing and regenerate the damaged tissue present in chronic obstructive pulmonary disease by means of their engraftment into the lung. Another approach could be the stimulation of resident stem cells by means of humoral factors or photobiostimulation. PMID:25548520

Sex differences in the pharmacokinetics of recombinant human granulocyte colony-stimulating factor in the rat.

PubMed

Tanaka, H; Kaneko, T

1991-01-01

The pharmacokinetics of recombinant human granulocyte colony-stimulating factor (rhG-CSF) were studied in male and female rats. The serum concentration of rhG-CSF after iv and sc administration to male and female Sprague-Dawley rats at a dose of 5 and 100 micrograms/kg was investigated by a sandwich enzyme-linked immunosorbent assay. After iv administration, AUC and half-lives of rhG-CSF in female rats were smaller than those for male rats. The volume of distribution of rhG-CSF in female rats was not significantly different from that in male rats. After sc administration, AUC, mean residence time, and half-lives of elimination phase in female rats were smaller than those for male rats. The in vitro biological activities of rhG-CSF were investigated using [3H]thymidine uptake assay in cultures of bone marrow cells obtained from male and female rat femur. Female rat bone marrow cells showed a similar dose-response profile to rhG-CSF to that of male rat bone marrow cells. The effect of rhG-CSF administration in rats was a specific activity on the neutrophil lineage with an increase of neutrophils in peripheral blood. The in vivo effects of rhG-CSF after iv and sc administration to male and female rats at 5 and 100 micrograms/kg doses were determined. After 100 micrograms/kg administration, the neutrophil count in female rats was similar to that in male rats in the early period; however, the neutrophil count in female rats was lower than that in male rats 24 hr after administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-LINGO-1 has no detectable immunomodulatory effects in preclinical and phase 1 studies

PubMed Central

Ranger, Ann; Ray, Soma; Szak, Suzanne; Dearth, Andrea; Allaire, Norm; Murray, Ronald; Gardner, Rebecca; Cadavid, Diego

2017-01-01

Objective: To evaluate whether the anti-LINGO-1 antibody has immunomodulatory effects. Methods: Human peripheral blood mononuclear cells (hPBMCs), rat splenocytes, and rat CD4+ T cells were assessed to determine whether LINGO-1 was expressed and was inducible. Anti-LINGO-1 Li81 (0.1–30 μg/mL) effect on proliferation/cytokine production was assessed in purified rat CD4+ T cells and hPBMCs stimulated with antibodies to CD3 +/– CD28. In humans, the effect of 2 opicinumab (anti-LINGO-1/BIIB033; 30, 60, and 100 mg/kg) or placebo IV administrations was evaluated in RNA from blood and CSF samples taken before and after administration in phase 1 clinical trials; paired samples were assessed for differentially expressed genes by microarray. RNA from human CSF cell pellets was analyzed by quantitative real-time PCR for changes in transcripts representative of cell types, activation markers, and soluble proteins of the adaptive/innate immune systems. ELISA quantitated the levels of CXCL13 protein in human CSF supernatants. Results: LINGO-1 is not expressed in hPBMCs, rat splenocytes, or rat CD4+ T cells; LINGO-1 blockade with Li81 did not affect T-cell proliferation or cytokine production from purified rat CD4+ T cells or hPBMCs. LINGO-1 blockade with opicinumab resulted in neither significant changes in immune system gene expression in blood and CSF, nor changes in CXCL13 CSF protein levels (clinical studies). Conclusions: These data support the hypothesis that LINGO-1 blockade does not affect immune function. Classification of evidence: This study provides Class II evidence that in patients with MS, opicinumab does not have immunomodulatory effects detected by changes in immune gene transcript expression. PMID:29259995

Escalated regeneration in sciatic nerve crush injury by the combined therapy of human amniotic fluid mesenchymal stem cells and fermented soybean extracts, Natto.

PubMed

Pan, Hung-Chuan; Yang, Dar-Yu; Ho, Shu-Peng; Sheu, Meei-Ling; Chen, Chung-Jung; Hwang, Shiaw-Min; Chang, Ming-Hong; Cheng, Fu-Chou

2009-08-23

Attenuation of inflammatory cell deposits and associated cytokines prevented the apoptosis of transplanted stem cells in a sciatic nerve crush injury model. Suppression of inflammatory cytokines by fermented soybean extracts (Natto) was also beneficial to nerve regeneration. In this study, the effect of Natto on transplanted human amniotic fluid mesenchymal stem cells (AFS) was evaluated. Peripheral nerve injury was induced in SD rats by crushing a sciatic nerve using a vessel clamp. Animals were categorized into four groups: Group I: no treatment; Group II: fed with Natto (16 mg/day for 7 consecutive days); Group III: AFS embedded in fibrin glue; Group IV: Combination of group II and III therapy. Transplanted AFS and Schwann cell apoptosis, inflammatory cell deposits and associated cytokines, motor function, and nerve regeneration were evaluated 7 or 28 days after injury. The deterioration of neurological function was attenuated by AFS, Natto, or the combined therapy. The combined therapy caused the most significantly beneficial effects. Administration of Natto suppressed the inflammatory responses and correlated with decreased AFS and Schwann cell apoptosis. The decreased AFS apoptosis was in line with neurological improvement such as expression of early regeneration marker of neurofilament and late markers of S-100 and decreased vacuole formation. Administration of either AFS, or Natto, or combined therapy augmented the nerve regeneration. In conclusion, administration of Natto may rescue the AFS and Schwann cells from apoptosis by suppressing the macrophage deposits, associated inflammatory cytokines, and fibrin deposits.

Ovarian membrane-type matrix metalloproteinases: induction of MMP14 and MMP16 during the periovulatory period in the rat, macaque, and human.

PubMed

Puttabyatappa, Muraly; Jacot, Terry A; Al-Alem, Linah F; Rosewell, Katherine L; Duffy, Diane M; Brännström, Mats; Curry, Thomas E

2014-08-01

An intrafollicular increase in proteolytic activity drives ovulatory events. Surprisingly, the periovulatory expression profile of the membrane-type matrix metalloproteinases (MT-MMPs), unique proteases anchored to the cell surface, has not been extensively examined. Expression profiles of the MT-MMPs were investigated in ovarian tissue from well-characterized rat and macaque periovulatory models and naturally cycling women across the periovulatory period. Among the six known MT-MMPs, mRNA expression of Mmp14, Mmp16, and Mmp25 was increased after human chorionic gonadotropin (hCG) administration in rats. In human granulosa cells, mRNA expression of MMP14 and MMP16 increased following hCG treatment. In contrast, mRNA levels of MMP16 and MMP25 in human theca cells were unchanged before ovulation but declined by the postovulatory stage. In macaque granulosa cells, hCG increased mRNA for MMP16 but not MMP14. Immunoblotting showed that protein levels of MMP14 and MMP16 in rats increased, similar to their mRNA expression. In macaque granulosa cells, only the active form of the MMP14 protein increased after hCG, unlike its mRNA or the proprotein. By immunohistochemistry, both MMP14 and MMP16 localized to the different ovarian cell types in rats and humans. Treatment with hCG resulted in intense immunoreactivity of MMP14 and MMP16 proteins in the granulosa and theca cells. The present study shows that MMP14 and MMP16 are increased by hCG administration in the ovulating follicle, demonstrating that these MMPs are conserved among rats, macaques, and humans. These findings suggest that MT-MMPs could have an important role in promoting ovulation and remodeling of the ovulated follicle into the corpus luteum. © 2014 by the Society for the Study of Reproduction, Inc.

Ovarian Membrane-Type Matrix Metalloproteinases: Induction of MMP14 and MMP16 During the Periovulatory Period in the Rat, Macaque, and Human1

PubMed Central

Puttabyatappa, Muraly; Jacot, Terry A.; Al-Alem, Linah F.; Rosewell, Katherine L.; Duffy, Diane M.; Brännström, Mats; Curry, Thomas E.

2014-01-01

ABSTRACT An intrafollicular increase in proteolytic activity drives ovulatory events. Surprisingly, the periovulatory expression profile of the membrane-type matrix metalloproteinases (MT-MMPs), unique proteases anchored to the cell surface, has not been extensively examined. Expression profiles of the MT-MMPs were investigated in ovarian tissue from well-characterized rat and macaque periovulatory models and naturally cycling women across the periovulatory period. Among the six known MT-MMPs, mRNA expression of Mmp14, Mmp16, and Mmp25 was increased after human chorionic gonadotropin (hCG) administration in rats. In human granulosa cells, mRNA expression of MMP14 and MMP16 increased following hCG treatment. In contrast, mRNA levels of MMP16 and MMP25 in human theca cells were unchanged before ovulation but declined by the postovulatory stage. In macaque granulosa cells, hCG increased mRNA for MMP16 but not MMP14. Immunoblotting showed that protein levels of MMP14 and MMP16 in rats increased, similar to their mRNA expression. In macaque granulosa cells, only the active form of the MMP14 protein increased after hCG, unlike its mRNA or the proprotein. By immunohistochemistry, both MMP14 and MMP16 localized to the different ovarian cell types in rats and humans. Treatment with hCG resulted in intense immunoreactivity of MMP14 and MMP16 proteins in the granulosa and theca cells. The present study shows that MMP14 and MMP16 are increased by hCG administration in the ovulating follicle, demonstrating that these MMPs are conserved among rats, macaques, and humans. These findings suggest that MT-MMPs could have an important role in promoting ovulation and remodeling of the ovulated follicle into the corpus luteum. PMID:24920038

Age-related increase in Wnt inhibitor causes a senescence-like phenotype in human cardiac stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakamura, Tamami; Hosoyama, Tohru; Regenerative Medicine Institute, Yamaguchi University Graduate School of Medicine

Aging of cardiac stem/progenitor cells (CSCs) impairs heart regeneration and leads to unsatisfactory outcomes of cell-based therapies. As the precise mechanisms underlying CSC aging remain unclear, the use of therapeutic strategies for elderly patients with heart failure is severely delayed. In this study, we used human cardiosphere-derived cells (CDCs), a subtype of CSC found in the postnatal heart, to identify secreted factor(s) associated with CSC aging. Human CDCs were isolated from heart failure patients of various ages (2–83 years old). Gene expression of key soluble factors was compared between CDCs derived from young and elderly patients. Among these factors, SFRP1,more » a gene encoding a Wnt antagonist, was significantly up-regulated in CDCs from elderly patients (≥65 years old). sFRP1 levels was increased significantly also in CDCs, whose senescent phenotype was induced by anti-cancer drug treatment. These results suggest the participation of sFRP1 in CSC aging. We show that the administration of recombinant sFRP1 induced cellular senescence in CDCs derived from young patients, as indicated by increased levels of markers such as p16, and a senescence-associated secretory phenotype. In addition, co-administration of recombinant sFRP1 could abrogate the accelerated CDC proliferation induced by Wnt3A. Taken together, our results suggest that canonical Wnt signaling and its antagonist, sFRP1, regulate proliferation of human CSCs. Furthermore, excess sFRP1 in elderly patients causes CSC aging. - Highlights: • Wnt signaling regulates proliferation of human cardiac stem cells. • Expression of sFRP1, which is a Wnt antagonist, is up-regulated in elderly patients with heart failure. • Expression of sFRP1 is increased in anti-cancer drug-induced senescent human cardiac stem cells. • sFRP1 causes cellular senescence of young patients-derived cardiac stem cells.« less

Oncolytic adenovirus that overproduces ADP and replicates selectively in tumors due to hTERT promoter-regulated E4 gene expression.

PubMed

Kuppuswamy, M; Spencer, J F; Doronin, K; Tollefson, A E; Wold, W S M; Toth, K

2005-11-01

We have constructed a novel oncolytic adenovirus (Ad) vector, named VRX-011, in which the replication of the vector is targeted to cancer cells by the replacement of the wild-type Ad E4 promoter with the human telomerase reverse transcriptase (hTERT) promoter. Genes in the Ad E4 transcription unit are essential for Ad replication; therefore, VRX-011 will grow efficiently only in cells in which the hTERT promoter is active, that is, in a wide range of cancer and immortalized cells but not in most somatic cells. Consistent with these expectations, VRX-011 replicated efficiently in all cancer cell lines examined, while its growth was restricted in various primary and normal cells. VRX-011 overexpresses ADP (also known as E3-11.6K), an Ad protein required for efficient cell lysis and release of virions from cells at late stages of infection. This overexpression enhances cell-to-cell spread and could significantly increase antitumor efficacy. In a xenograft model in nude mice, both intratumoral and intravenous administration of VRX-011 effectively suppressed the growth of subcutaneous Hep3B human liver tumors. Also, intravenous delivery of VRX-011 greatly reduced the number and size of A549 human lung cancer cell nodules in a disseminated lung tumor model in nude mice. Importantly, tail vein administration of different doses of VRX-011 in C57BL/6 mice showed minimal liver toxicity. Considering its broad range of lytic replication in cancer cells, its attenuated phenotype in primary cells, its efficacy in suppressing xenografts, and its low toxicity in mouse liver, VRX-011 is a promising candidate for further evaluation as an anticancer therapeutic.

21 CFR 1271.150 - Current good tissue practice requirements.

Code of Federal Regulations, 2011 CFR

2011-04-01

....150 Section 1271.150 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS Current Good Tissue Practice § 1271.150... implemented for reproductive HCT/Ps described in § 1271.10 and regulated solely under section 361 of the...

21 CFR 1271.150 - Current good tissue practice requirements.

Code of Federal Regulations, 2012 CFR

2012-04-01

....150 Section 1271.150 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS Current Good Tissue Practice § 1271.150... implemented for reproductive HCT/Ps described in § 1271.10 and regulated solely under section 361 of the...

21 CFR 1271.150 - Current good tissue practice requirements.

Code of Federal Regulations, 2013 CFR

2013-04-01

....150 Section 1271.150 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS Current Good Tissue Practice § 1271.150... implemented for reproductive HCT/Ps described in § 1271.10 and regulated solely under section 361 of the...

21 CFR 1271.150 - Current good tissue practice requirements.

Code of Federal Regulations, 2014 CFR

2014-04-01

....150 Section 1271.150 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS Current Good Tissue Practice § 1271.150... implemented for reproductive HCT/Ps described in § 1271.10 and regulated solely under section 361 of the...

21 CFR 1271.45 - What requirements does this subpart contain?

Code of Federal Regulations, 2011 CFR

2011-04-01

....45 Section 1271.45 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS Donor Eligibility § 1271.45 What... oocyte donor and the semen donor. (c) Prohibition on use. An HCT/P must not be implanted, transplanted...

Schedule-dependent cytotoxic synergism of pemetrexed and erlotinib in BXPC-3 and PANC-1 human pancreatic cancer cells.

PubMed

Wang, Lin; Zhu, Zhi-Xia; Zhang, Wen-Ying; Zhang, Wei-Min

2011-09-01

Previous studies have shown that both pemetrexed, a cytotoxic drug, and erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), inhibit the cell growth of pancreatic cancer cells. However, whether they exert a synergistic antitumor effect on pancreatic cancer cells remains unknown. The present study aimed to assess the synergistic effect of erlotinib in combination with pemetrexed using different sequential administration schedules on the proliferation of human pancreatic cancer BXPC-3 and PANC-1 cells and to probe its cellular mechanism. The EGFR and K-ras gene mutation status was examined by quantitative PCR high-resolution melting (qPCR-HRM) analysis. BXPC-3 and PANC-1 cells were incubated with pemetrexed and erlotinib using different administration schedules. MTT assay was used to determine cytotoxicity, and cell cycle distribution was determined by flow cytometry. The expression and phosphorylation of EGFR, HER3, AKT and MET were determined using Western blotting. Both pemetrexed and erlotinib inhibited the proliferation of BXPC-3 and PANC-1 cells in a dose- and time-dependent manner in vitro. Synergistic effects on cell proliferation were observed when pemetrexed was used in combination with erlotinib. The degree of the synergistic effects depended on the administration sequence, which was most obvious when erlotinib was sequentially administered at 24-h interval following pemetrexed. Cell cycle studies revealed that pemetrexed induced S arrest and erlotinib induced G(0)/G(1) arrest. The sequential administration of erlotinib following pemetrexed induced S arrest. Western blot analyses showed that pemetrexed increased and erlotinib decreased the phosphorylation of EGFR, HER3 and AKT, respectively. However, both pemetrexed and erlotinib exerted no significant effects on the phosphorylation of c-MET. The phosphorylation of EGFR, HER3 and AKT was significantly suppressed by scheduled incubation with pemetrexed followed by erlotinib, but not by concomitant or sequential incubation with erlotinib followed by pemetrexed. In summary, our results demonstrated that the combined use of erlotinib and pemetrexed exhibited a strong synergism in BXPC-3 and PANC-1 cells. The inhibitory effects were strongest after sequential administration of pemetrexed followed by erlotinib. The synergistic effects may be related to activation of the EGFR/HER3/AKT pathway induced by pemetrexed.

21 CFR 1271.65 - How do I store an HCT/P from a donor determined to be ineligible, and what uses of the HCT/P are...

Code of Federal Regulations, 2010 CFR

2010-04-01

... CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND... first-degree or second-degree blood relative; (ii) The HCT/P consists of reproductive cells or tissue...

21 CFR 1271.65 - How do I store an HCT/P from a donor determined to be ineligible, and what uses of the HCT/P are...

Code of Federal Regulations, 2014 CFR

2014-04-01

... CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND... first-degree or second-degree blood relative; (ii) The HCT/P consists of reproductive cells or tissue...

21 CFR 1271.65 - How do I store an HCT/P from a donor determined to be ineligible, and what uses of the HCT/P are...

Code of Federal Regulations, 2011 CFR

2011-04-01

... CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND... first-degree or second-degree blood relative; (ii) The HCT/P consists of reproductive cells or tissue...

Human melanoma immunotherapy using tumor antigen-specific T cells generated in humanized mice

PubMed Central

Hu, Zheng; Xia, Jinxing; Fan, Wei; Wargo, Jennifer; Yang, Yong-Guang

2016-01-01

A major factor hindering the exploration of adoptive immunotherapy in preclinical settings is the limited availability of tumor-reactive human T cells. Here we developed a humanized mouse model that permits large-scale production of human T cells expressing the engineered melanoma antigen MART-1-specific TCR. Humanized mice, made by transplantation of human fetal thymic tissue and CD34+ cells virally-transduced with HLA class I-restricted melanoma antigen (MART-1)-specific TCR gene, showed efficient development of MART-1-TCR+ human T cells with predominantly CD8+ cells. Importantly, MART-1-TCR+CD8+ T cells developing in these mice were capable of mounting antigen-specific responses in vivo, as evidenced by their proliferation, phenotypic conversion and IFN-γ production following MART-1 peptide immunization. Moreover, these MART-1-TCR+CD8+ T cells mediated efficient killing of melanoma cells in an HLA/antigen-dependent manner. Adoptive transfer of in vitro expanded MART-1-TCR+CD8+ T cells induced potent antitumor responses that were further enhanced by IL-15 treatment in melanoma-bearing recipients. Finally, a short incubation of MART-1-specific T cells with rapamycin acted synergistically with IL-15, leading to significantly improved tumor-free survival in recipients with metastatic melanoma. These data demonstrate the practicality of using humanized mice to produce potentially unlimited source of tumor-specific human T cells for experimental and preclinical exploration of cancer immunotherapy. This study also suggests that pretreatment of tumor-reactive T cells with rapamycin in combination with IL-15 administration may be a novel strategy to improve the efficacy of adoptive T cell therapy. PMID:26824989

Administration of recombinant human granulocyte-colony-stimulating factor does not induce long-lasting detectable epigenetic alterations in healthy donors.

PubMed

Leitner, Gerda C; Faschingbauer, Martin; Wenda, Sabine; Weigel, Günter; Fischer, Gottfried

2014-12-01

The short-term safety profile of recombinant human granulocyte-colony-stimulating factor (rHuG-CSF) in the allogeneic stem cell setting seems acceptable; only few data on long-term safety are available. To further study possible epigenetic alterations, we investigated prospectively the influence of rHuG-CSF on DNA methyltransferase (DNMT) activity and on changes in DNA methylation of candidate genes in peripheral blood cells of healthy unrelated stem cell donors within an observation period of 1 year. In this study, 20 stem cell donors (14 male/six female; median age, 40 years; range, 22-54 years) and 20 sex- and age-matched blood component donors (controls) were included. Sampling was performed before rHuG-CSF administration; at the time of donation; and on Days (+1), 7, 30, 100, 180, and 360 in both groups. Analysis of DNMT activity in nuclear extracts was performed using a modified radionuclide assay. We performed methylation-specific polymerase chain reaction to detect the methylation status of promoter CpG islands of the genes of the retinoic acid receptor beta (RAR-B) and the Ras association domain family 1A (RASSF1A). DNMT activity increased significantly on the day of donation and 1 day after (p < 0.05). By Day +7 baseline values were reached. No further significant alterations of DNMT activity in the treated group compared to the controls were observed. We could not detect any differences in the gene methylation of RAR-B and RASSF1A between both groups. In our prospective study no evidence of long-lasting increased DNMT activity or enhanced DNA methylation in a limited panel of target genes after recombinant human G-CSF administration was observed in healthy stem cell donors. © 2014 AABB.

21 CFR 1271.210 - Supplies and reagents.

Code of Federal Regulations, 2010 CFR

2010-04-01

...) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES... spread of communicable diseases. Verification may be accomplished by the establishment that uses the...

21 CFR 1271.90 - Are there exceptions from the requirement of determining donor eligibility, and what labeling...

Code of Federal Regulations, 2010 CFR

2010-04-01

... CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND... donor screening or testing under §§ 1271.75, 1271.80 and 1271.85 for: (1) Cells and tissues for autologous use; or (2) Reproductive cells or tissue donated by a sexually intimate partner of the recipient...

21 CFR 1271.90 - Are there exceptions from the requirement of determining donor eligibility, and what labeling...

Code of Federal Regulations, 2014 CFR

2014-04-01

... CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND... donor screening or testing under §§ 1271.75, 1271.80 and 1271.85 for: (1) Cells and tissues for autologous use; or (2) Reproductive cells or tissue donated by a sexually intimate partner of the recipient...

Emodin induces apoptosis of human osteosarcoma cells via mitochondria- and endoplasmic reticulum stress-related pathways.

PubMed

Ying, Jinhe; Xu, Huan; Wu, Dhua; Wu, Xiaoguang

2015-01-01

Emodin showed anti-cancer activity against multiple human malignant tumors by inducing apoptosis. However, the apoptotic inducing effect against human osteosarcoma and related mechanism are still not studied. This study was aimed to investigate them. Emodin was used to incubate human OS cell U2OS cells at serially diluted concentrations. Hoechst staining was used to evaluate apoptosis; flow cytometry was applied to assess the collapse of mitochondrial membrane potential (MMP); intracellular ROS generation was detected by DCFH-DA staining; endoplasmic reticulum stress activation was examined by western blotting. Cell apoptosis of U2OS cells was induced by emodin incubation in a concentration-dependent manner; MMP collapse and ROS generation were identified at starting concentration of 80 μmol/L of emodin in a concentration-dependent manner. ER stress activation was found at beginning concentration of 40 μmol/L of emodin. The MMP collapse was inhibited while the ER stress was not inhibited by NAC administration. Emodin induces death of human osteosarcoma cells by initiating ROS-dependent mitochondria-induced and ROS-independent ER stress-induced apoptosis.

Emodin induces apoptosis of human osteosarcoma cells via mitochondria- and endoplasmic reticulum stress-related pathways

PubMed Central

Ying, Jinhe; Xu, Huan; Wu, Dhua; Wu, Xiaoguang

2015-01-01

Aim: Emodin showed anti-cancer activity against multiple human malignant tumors by inducing apoptosis. However, the apoptotic inducing effect against human osteosarcoma and related mechanism are still not studied. This study was aimed to investigate them. Methods: Emodin was used to incubate human OS cell U2OS cells at serially diluted concentrations. Hoechst staining was used to evaluate apoptosis; flow cytometry was applied to assess the collapse of mitochondrial membrane potential (MMP); intracellular ROS generation was detected by DCFH-DA staining; endoplasmic reticulum stress activation was examined by western blotting. Results: Cell apoptosis of U2OS cells was induced by emodin incubation in a concentration-dependent manner; MMP collapse and ROS generation were identified at starting concentration of 80 μmol/L of emodin in a concentration-dependent manner. ER stress activation was found at beginning concentration of 40 μmol/L of emodin. The MMP collapse was inhibited while the ER stress was not inhibited by NAC administration. Conclusions: Emodin induces death of human osteosarcoma cells by initiating ROS-dependent mitochondria-induced and ROS-independent ER stress-induced apoptosis. PMID:26722474

21 CFR 1271.180 - Procedures.

Code of Federal Regulations, 2010 CFR

2010-04-01

... UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND... organization, you must verify that the procedures meet the requirements of this part and are appropriate for...

Pharmacokinetic and pharmacodynamic comparisons between human granulocyte colony-stimulating factor purified from human bladder carcinoma cell line 5637 culture medium and recombinant human granulocyte colony-stimulating factor produced in Escherichia coli.

PubMed

Tanaka, H; Kaneko, T

1992-07-01

The pharmacokinetics and biological activities of recombinant human granulocyte colony-stimulating factor (hG-CSF) produced in Escherichia coli were compared with those of hG-CSF purified from human bladder carcinoma cell line 5637 culture medium (5637-hG-CSF). Recombinant hG-CSF was biologically active in a bone marrow cell proliferation assay in vitro, with a dose-response curve similar to that of 5637-hG-CSF. The effects of 5637- and recombinant hG-CSF administered via i.v. injection to rats showed similar response patterns of neutrophil counts in peripheral blood. From these results, it is concluded that the O-linked sugar chain of hG-CSF does not contribute to the in vitro and in vivo biological activities. The pharmacokinetics of both forms of hG-CSF in rats were investigated using a sandwich enzyme-linked immunosorbent assay. After i.v. administration, the serum concentration-time curves of 5637- and recombinant hG-CSF declined biexponentially. Total body clearance and steady-state volume of distribution of 5637-hG-CSF were smaller than those for the recombinant form. After s.c. administration, a lower peak serum level, smaller AUC, and lower bioavailability of 5637-hG-CSF were observed compared to recombinant hG-CSF.

AMPK/p53 Axis Is Essential for α-Lipoic Acid-Regulated Metastasis in Human and Mouse Colon Cancer Cells.

PubMed

Park, Sunmi; Choi, Seung Kug; Choi, Yura; Moon, Hyun-Seuk

2015-10-01

α-Lipoic acid (ALA) has an anticancer property of lung, cervix, and prostate cancer cells. However, direct evidence that ALA contributes to the development of colon cancer has not been fully elucidated. In addition, no previous studies have evaluated whether ALA may regulate malignant potential, such as adhesion, invasion, and colony formation of colon cancer cells. To address the aforementioned questions, we conducted in vitro ALA signaling studies using human (HT29) and mouse (MCA38) colon cancer cell lines. We observed that cell proliferation is reduced by ALA administration in a dose-dependent manner in human and mouse colon cancer cell lines. Specifically, 0.5 to 1 mM concentration of ALA significantly decreased cell proliferation when compared with control. Similarly, we found that ALA downregulates adhesion, invasion, and colony formation. Finally, we observed that ALA activates p53 and AMPK signaling pathways in human and mouse colon cancer cells. We found for the first time that ALA suppresses cell proliferation and malignant potential via p53 and AMPK signaling pathways in human and mouse colon cancer cells. These new and early mechanistic studies provide a causal role of ALA in colon cancer, suggesting that ALA might be a useful agent in the management or chemoprevention of colon cancer.

The novel JNK inhibitor AS602801 inhibits cancer stem cells in vitro and in vivo.

PubMed

Okada, Masashi; Kuramoto, Kenta; Takeda, Hiroyuki; Watarai, Hikaru; Sakaki, Hirotsugu; Seino, Shizuka; Seino, Manabu; Suzuki, Shuhei; Kitanaka, Chifumi

2016-05-10

A phase 2 clinical trial investigating the efficacy and safety of AS602801, a newly developed JNK inhibitor, in the treatment of inflammatory endometriosis is complete. We are now examining whether AS602801 acts against human cancer cells in vitro and in vivo. In vitro, AS602801 exhibited cytotoxicity against both serum-cultured non-stem cancer cells and cancer stem cells derived from human pancreatic cancer, non-small cell lung cancer, ovarian cancer and glioblastoma at concentrations that did not decrease the viability of normal human fibroblasts. AS602801 also inhibited the self-renewal and tumor-initiating capacity of cancer stem cells surviving AS602801 treatment. Cancer stem cells in established xenograft tumors were reduced by systemic administration of AS602801 at a dose and schedule that did not adversely affect the health of the tumor-bearing mice. These findings suggest AS602801 is a promising anti-cancer stem cell agent, and further investigation of the utility of AS602801 in the treatment of cancer seems warranted.

Comparison of intracellular accumulation and cytotoxicity of free mTHPC and mTHPC-loaded PLGA nanoparticles in human colon carcinoma cells.

PubMed

Löw, Karin; Knobloch, Thomas; Wagner, Sylvia; Wiehe, Arno; Engel, Andrea; Langer, Klaus; von Briesen, Hagen

2011-06-17

The second generation photosensitizer mTHPC was approved by the European Medicines Agency (EMA) for the palliative treatment of advanced head and neck cancer in October 2001. It is known that mTHPC possesses a significant phototoxicity against a variety of human cancer cells in vitro but also exhibits dark toxicity and can cause adverse effects (especially skin photosensitization). Due to its poor water solubility, the administration of hydrophobic photosensitizer still presents several difficulties. To overcome the administration problems, the use of nanoparticles as drug carrier systems is much investigated. Nanoparticles based on poly(lactic-co-glycolic acid) (PLGA) have been extensively studied as delivery systems into tumours due to their biocompatibility and biodegradability. The goal of this study was the comparison of free mTHPC and mTHPC-loaded PLGA nanoparticles concerning cytotoxicity and intracellular accumulation in human colon carcinoma cells (HT29). The nanoparticles delivered the photosensitizer to the colon carcinoma cells and enabled drug release without losing its activity. The cytotoxicity assays showed a time- and concentration-dependent decrease in cell proliferation and viability after illumination. However, first and foremost mTHPC lost its dark toxic effects using the PLGA nanoparticles as a drug carrier system. Therefore, PLGA nanoparticles are a promising drug carrier system for the hydrophobic photosensitizer mTHPC.

Effects of pegylated recombinant human megakaryocyte growth and development factor on thrombocytopenia induced by a new myelosuppressive chemotherapy regimen in mice.

PubMed

Akahori, H; Shibuya, K; Ozai, M; Ida, M; Kabaya, K; Kato, T; Miyazaki, H

1996-11-01

Thrombopoietin, the endogenous c-Mpl ligand, is a novel lineage-specific hematopoietic factor that plays a pivotal role in the regulation of megakaryocytopoiesis and thrombopoiesis. In this study, we examined the effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), a truncated molecule of recombinant human c-Mpl ligand derivatized with polyethylene glycol, on myelosuppressive chemotherapy-induced thrombocytopenia in mice. We developed a new murine model of thrombocytopenia induced by i.v. injections of mitomycin C (MMC) for two consecutive days. In control mice, platelet counts began to decrease on day 6, reached a nadir of less than 5% of basal level on day 14, and could not recover to basal level by day 26. Administration of PEG-rHuMGDF greatly enhanced recovery of the number of megakaryocyte progenitor cells and the megakaryocytes in bone marrow, and markedly reduced the severity of thrombocytopenia; it also accelerated platelet recovery in a dose-dependent manner in myelosuppressed mice. Mice receiving consecutive administration of higher doses of PEG-rHuMGDF showed no thrombocytopenia but rather had platelet counts being increased over basal level. Although absolute neutrophil counts and red cell counts also were decreased following MMC treatment, administration of PEG-rHuMGDF also improved neutropenia and anemia. Administration of PEG-rHuMGDF on alternate days or once a week after chemotherapy was almost as effective as consecutive administration in improving thrombocytopenia. Combined administration of PEG-rHuMGDF and rHuG-CSF had an additive effect on improvement of thrombocytopenia and neutropenia. These results suggest that PEG-rHuMGDF is a therapeutically effective agent in the treatment of thrombocytopenia associated with chemotherapy.

Autologous cell therapy for cisplatin-induced acute kidney injury by using non-expanded adipose tissue-derived cells.

PubMed

Yasuda, Kaoru; Ozaki, Takenori; Saka, Yousuke; Yamamoto, Tokunori; Gotoh, Momokazu; Ito, Yasuhiko; Yuzawa, Yukio; Matsuo, Seiichi; Maruyama, Shoichi

2012-10-01

Recent studies have demonstrated that cultured mesenchymal stromal cells derived from adipose tissue are useful for regenerative cell therapy. The stromal vascular fraction (SVF) can be obtained readily without culturing and may be clinically applicable. We investigated the therapeutic effects of SVF and used it in the treatment of acute kidney injury (AKI). Liposuction aspirates were obtained from healthy donors who had provided written informed consent. We harvested the SVF and determined the growth factor secretion and anti-apoptotic ability with conditioned medium. To investigate the effect of SVF on AKI, cisplatin was injected into rats and SVF was administrated into the subcupsula of the kidney. Both human and rat SVF cells secreted vascular endothelial growth factor-A (VEGF) and hepatocyte growth factor (HGF). Human SVF-conditioned media had an anti-apoptotic effect, which was inhibited by anti-HGF antibody (Ab) but not by anti-VEGF Ab. In vivo, SVF significantly ameliorated renal function, attenuated tubular damage and increased the cortical blood flow speed. In the SVF-treated group, VEGF levels in the cortex and HGF levels in both the cortex and medulla, especially tubules in the medulla, were significantly higher. Immunostaining revealed that SVF cells expressing VEGF and HGF and remained in the subcapsule on day 14. The present study demonstrates that a subcapsular injection of non-expanded SVF cells ameliorates rat AKI, and that the mechanism probably involves secretion of renoprotective molecules. Administration of human SVF may be clinically applicable and useful as a novel autologous cell therapy against kidney diseases.

21 CFR 640.13 - Collection of the blood.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Collection of the blood. 640.13 Section 640.13 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.13 Collection of the...

21 CFR 640.13 - Collection of the blood.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Collection of the blood. 640.13 Section 640.13 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.13 Collection of the...

21 CFR 640.13 - Collection of the blood.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Collection of the blood. 640.13 Section 640.13 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.13 Collection of the blood. (a...

21 CFR 640.13 - Collection of the blood.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Collection of the blood. 640.13 Section 640.13 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.13 Collection of the...

21 CFR 640.13 - Collection of the blood.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Collection of the blood. 640.13 Section 640.13 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.13 Collection of the...

21 CFR 640.17 - Modifications for specific products.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Modifications for specific products. 640.17 Section 640.17 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.17 Modifications for...

Human lacrimal gland regeneration: Perspectives and review of literature

PubMed Central

Tiwari, Shubha; Ali, Mohammad Javed; Vemuganti, Geeta K.

2013-01-01

The human lacrimal gland is an essential component of the lacrimal functional unit (LFU). Any perturbation of this unit can lead to the debilitating morbid condition called the dry eye syndrome (DES). The current line of therapy available for dry eye remains supportive and palliative with the patient being dependent on life long and frequent administration of lubricating eye drops. Even advanced therapies like punctual plugs, cyclosporine B administration, and salivary gland auto-transplantation have led to a limited success. Under these scenarios, the option of cell based therapy needs to be explored to provide better and long term relief to these patients. This review gives an overview of the efforts in lacrimal gland regeneration and examines the past and ongoing research in cell based therapies in animals as well as human lacrimal gland cultures. The authors discuss their first of its kind functionally viable human lacrimal gland in vitro culture system from fresh exenteration specimens. A brief overview of research in near future and the potential implications of lacrimal gland regenerative therapies have been discussed. PMID:24526853

Human lacrimal gland regeneration: Perspectives and review of literature.

PubMed

Tiwari, Shubha; Ali, Mohammad Javed; Vemuganti, Geeta K

2014-01-01

The human lacrimal gland is an essential component of the lacrimal functional unit (LFU). Any perturbation of this unit can lead to the debilitating morbid condition called the dry eye syndrome (DES). The current line of therapy available for dry eye remains supportive and palliative with the patient being dependent on life long and frequent administration of lubricating eye drops. Even advanced therapies like punctual plugs, cyclosporine B administration, and salivary gland auto-transplantation have led to a limited success. Under these scenarios, the option of cell based therapy needs to be explored to provide better and long term relief to these patients. This review gives an overview of the efforts in lacrimal gland regeneration and examines the past and ongoing research in cell based therapies in animals as well as human lacrimal gland cultures. The authors discuss their first of its kind functionally viable human lacrimal gland in vitro culture system from fresh exenteration specimens. A brief overview of research in near future and the potential implications of lacrimal gland regenerative therapies have been discussed.

Human Hepatocyte Growth Factor (hHGF)-Modified Hepatic Oval Cells Improve Liver Transplant Survival

PubMed Central

Li, Li; Ran, Jiang-Hua; Li, Xue-Hua; Liu, Zhi-Heng; Liu, Gui-Jie; Gao, Yan-Chao; Zhang, Xue-Li; Sun, Hiu-Dong

2012-01-01

Despite progress in the field of immunosuppression, acute rejection is still a common postoperative complication following liver transplantation. This study aims to investigate the capacity of the human hepatocyte growth factor (hHGF) in modifying hepatic oval cells (HOCs) administered simultaneously with orthotopic liver transplantation as a means of improving graft survival. HOCs were activated and isolated using a modified 2-acetylaminofluorene/partial hepatectomy (2-AAF/PH) model in male Lewis rats. A HOC line stably expressing the HGF gene was established following stable transfection of the pBLAST2-hHGF plasmid. Our results demonstrated that hHGF-modified HOCs could efficiently differentiate into hepatocytes and bile duct epithelial cells in vitro. Administration of HOCs at the time of liver transplantation induced a wider distribution of SRY-positive donor cells in liver tissues. Administration of hHGF-HOC at the time of transplantation remarkably prolonged the median survival time and improved liver function for recipients compared to these parameters in the other treatment groups (P<0.05). Moreover, hHGF-HOC administration at the time of liver transplantation significantly suppressed elevation of interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) levels while increasing the production of IL-10 and TGF-β1 (P<0.05). HOC or hHGF-HOC administration promoted cell proliferation, reduced cell apoptosis, and decreased liver allograft rejection rates. Furthermore, hHGF-modified HOCs more efficiently reduced acute allograft rejection (P<0.05 versus HOC transplantation only). Our results indicate that the combination of hHGF-modified HOCs with liver transplantation decreased host anti-graft immune responses resulting in a reduction of allograft rejection rates and prolonging graft survival in recipient rats. This suggests that HOC-based cell transplantation therapies can be developed as a means of treating severe liver injuries. PMID:23028627

Augmentation of DHCR24 expression by hepatitis C virus infection facilitates viral replication in hepatocytes.

PubMed

Takano, Takashi; Tsukiyama-Kohara, Kyoko; Hayashi, Masahiro; Hirata, Yuichi; Satoh, Masaaki; Tokunaga, Yuko; Tateno, Chise; Hayashi, Yukiko; Hishima, Tsunekazu; Funata, Nobuaki; Sudoh, Masayuki; Kohara, Michinori

2011-09-01

We characterized the role of 24-dehydrocholesterol reductase (DHCR24) in hepatitis C virus infection (HCV). DHCR24 is a cholesterol biosynthetic enzyme and cholesterol is a major component of lipid rafts, which is reported to play an important role in HCV replication. Therefore, we examined the potential of DHCR24 as a target for novel HCV therapeutic agents. We examined DHCR24 expression in human hepatocytes in both the livers of HCV-infected patients and those of chimeric mice with human hepatocytes. We targeted DHCR24 with siRNA and U18666A which is an inhibitor of both DHCR24 and cholesterol synthesis. We measured the level of HCV replication in these HCV replicon cell lines and HCV infected cells. U18666A was administrated into chimeric mice with humanized liver, and anti-viral effects were assessed. Expression of DHCR24 was induced by HCV infection in human hepatocytes in vitro, and in human hepatocytes of chimeric mouse liver. Silencing of DHCR24 by siRNA decreased HCV replication in replicon cell lines and HCV JFH-1 strain-infected cells. Treatment with U18666A suppressed HCV replication in the replicon cell lines. Moreover, to evaluate the anti-viral effect of U18666A in vivo, we administrated U18666A with or without pegylated interferon to chimeric mice and observed an inhibitory effect of U18666A on HCV infection and a synergistic effect with interferon. DHCR24 is an essential host factor which augmented its expression by HCV infection, and plays a significant role in HCV replication. DHCR24 may serve as a novel anti-HCV drug target. Copyright © 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Histologic evaluation of human benign prostatic hyperplasia treated by dutasteride: a study by xenograft model with improved severe combined immunodeficient mice.

PubMed

Tsujimura, Akira; Fukuhara, Shinichiro; Soda, Tetsuji; Takezawa, Kentaro; Kiuchi, Hiroshi; Takao, Tetsuya; Miyagawa, Yasushi; Nonomura, Norio; Adachi, Shigeki; Tokita, Yoriko; Nomura, Taisei

2015-01-01

To evaluate histologic change in human prostate samples treated with dutasteride and to elucidate direct effects of dutasteride on human prostate tissue, the present study was conducted by using a xenograft model with improved severe combined immunodeficient (super-SCID) mice, although it is well known that dutasteride reduces prostate volume. After establishment of a xenograft model of human benign prostatic hyperplasia in morphology and function, samples implanted into super-SCID mice with and without dutasteride were evaluated pathohistologically at 2 and 6 months after initiation of dutasteride administration. The proliferative index evaluated by Ki-67 staining was significantly lower in the dutasteride group than the control at 2 and 6 months after administration. Apoptotic index evaluated by the terminal transferase TdT-mediated dUTP-biotin nick end labeling staining was higher in the dutasteride group than the control at 2 and 6 months after administration. Quick scores in the dutasteride group for staining of both cyclooxygenase-2 (Cox-2) and Ras homolog gene family, member A (RhoA) were significantly lower than those in the control group at 2 and 6 months after administration. Dutasteride inhibits cell proliferation and induces apoptosis of prostatic cells, causing a reduced prostate volume. Furthermore, decreased expression of Cox-2 and RhoA within benign prostatic hyperplasia tissue by dutasteride may induce an early effect on improvement of lower urinary tract symptoms, probably by attenuating inflammation reaction of the prostate and decreasing intraurethral pressure, other than the mechanism of reduced prostate volume. Copyright © 2015 Elsevier Inc. All rights reserved.

Immune Humanization of Immunodeficient Mice Using Diagnostic Bone Marrow Aspirates from Carcinoma Patients

PubMed Central

Werner-Klein, Melanie; Proske, Judith; Werno, Christian; Schneider, Katharina; Hofmann, Hans-Stefan; Rack, Brigitte; Buchholz, Stefan; Ganzer, Roman; Blana, Andreas; Seelbach-Göbel, Birgit; Nitsche, Ulrich

2014-01-01

Tumor xenografts in immunodeficient mice, while routinely used in cancer research, preclude studying interactions of immune and cancer cells or, if humanized by allogeneic immune cells, are of limited use for tumor-immunological questions. Here, we explore a novel way to generate cancer models with an autologous humanized immune system. We demonstrate that hematopoietic stem and progenitor cells (HSPCs) from bone marrow aspirates of non-metastasized carcinoma patients, which are taken at specialized centers for diagnostic purposes, can be used to generate a human immune system in NOD-scid IL2rγ(null) (NSG) and HLA-I expressing NSG mice (NSG-HLA-A2/HHD) comprising both, lymphoid and myeloid cell lineages. Using NSG-HLA-A2/HHD mice, we show that responsive and self-tolerant human T cells develop and human antigen presenting cells can activate human T cells. As critical factors we identified the low potential of bone marrow HSPCs to engraft, generally low HSPC numbers in patient-derived bone marrow samples, cryopreservation and routes of cell administration. We provide here an optimized protocol that uses a minimum number of HSPCs, preselects high-quality bone marrow samples defined by the number of initially isolated leukocytes and intra-femoral or intra-venous injection. In conclusion, the use of diagnostic bone marrow aspirates from non-metastasized carcinoma patients for the immunological humanization of immunodeficient mice is feasible and opens the chance for individualized analyses of anti-tumoral T cell responses. PMID:24830425

Identification, characterization and potent antitumor activity of ECO-4601, a novel peripheral benzodiazepine receptor ligand.

PubMed

Gourdeau, Henriette; McAlpine, James B; Ranger, Maxime; Simard, Bryan; Berger, Francois; Beaudry, Francis; Farnet, Chris M; Falardeau, Pierre

2008-05-01

ECO-4601 is a structurally novel farnesylated dibenzodiazepinone discovered through DECIPHER technology, Thallion's proprietary drug discovery platform. The compound was shown to have a broad cytotoxic activity in the low micromolar range when tested in the NCI 60 cell line panel. In the work presented here, ECO-4601 was further evaluated against brain tumor cell lines. Preliminary mechanistic studies as well as in vivo antitumor evaluation were performed. Since ECO-4601 has a benzodiazepinone moiety, we first investigated if it binds the central and/or peripheral benzodiazepine receptors. ECO-4601 was tested in radioligand binding assays on benzodiazepine receptors obtained from rat hearts. The ability of ECO-4601 to inhibit the growth of CNS cancers was evaluated on a panel of mouse, rat and human glioma cell lines using a standard MTT assay. Antitumor efficacy studies were performed on gliomas (rat and human), human breast and human prostate mouse tumor xenografts. Antitumor activity and pharmacokinetic analysis of ECO-4601 was evaluated following intravenous (i.v.), subcutaneous (s.c.), and intraperitoneal (i.p.) bolus administrations. ECO-4601 was shown to bind the peripheral but not the central benzodiazepine receptor and inhibited the growth of CNS tumor cell lines. Bolus s.c. and i.p. administration gave rise to low but sustained drug exposure, and resulted in moderate to significant antitumor activity at doses that were well tolerated. In a rat glioma (C6) xenograft model, ECO-4601 produced up to 70% tumor growth inhibition (TGI) while in a human glioma (U-87MG) xenograft, TGI was 34%. Antitumor activity was highly significant in both human hormone-independent breast (MDA-MB-231) and prostate (PC-3) xenografts, resulting in TGI of 72 and 100%, respectively. On the other hand, i.v. dosing was followed by rapid elimination of the drug and was ineffective. Antitumor efficacy of ECO-4601 appears to be associated with the exposure parameter AUC and/or sustained drug levels rather than C (max). These in vivo data constitute a rationale for clinical studies testing prolonged continuous administration of ECO-4601.

Dietary phenethyl isothiocyanate inhibition of androgen-responsive LNCaP prostate cancer cell tumor growth correlates with decreased angiogenesis

USDA-ARS?s Scientific Manuscript database

Phenethyl isothiocyanate (PEITC), found in certain cruciferous vegetables, has antitumor activity in several cancer models, including prostate cancer. In our xenograft model, dietary administration of PEITC (100-150 mg/kg/d) inhibited androgen-responsive LNCaP human prostate cancer cell tumor growth...

21 CFR 1271.10 - Are my HCT/P's regulated solely under section 361 of the PHS Act and the regulations in this part...

Code of Federal Regulations, 2010 CFR

2010-04-01

...) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES... objective intent; (3) The manufacture of the HCT/P does not involve the combination of the cells or tissues... not have a systemic effect and is not dependent upon the metabolic activity of living cells for its...

Human umbilical cord blood-derived mesenchymal stem cells attenuate hyperoxia-induced lung injury in neonatal rats.

PubMed

Chang, Yun Sil; Oh, Wonil; Choi, Soo Jin; Sung, Dong Kyung; Kim, Soo Yoon; Choi, Eun Yang; Kang, Saem; Jin, Hye Jin; Yang, Yoon Sun; Park, Won Soon

2009-01-01

Recent evidence suggests mesenchymal stem cells (MSCs) can downmodulate bleomycin-induced lung injury, and umbilical cord blood (UCB) is a promising source for human MSCs. This study examined whether intratracheal or intraperitoneal transplantation of human UCB-derived MSCs can attenuate hyperoxia-induced lung injury in immunocompetent newborn rats. Wild-type Sprague-Dawley rats were randomly exposed to 95% oxygen or air from birth. In the transplantation groups, a single dose of PKH26-labeled human UCB-derived MSCs was administered either intratracheally (2 x 10(6) cells) or intraperitoneally (5 x 10(5) cells) at postnatal day (P) 5. At P14, the harvested lungs were examined for morphometric analyses of alveolarization and TUNEL staining, as well as the myeoloperoxidase activity, the level of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and transforming growth factor (TGF)-beta mRNA, alpha-smooth muscle actin (SMA) protein, and collagen levels. Differentiation of MSCs to the respiratory epithelium was also evaluated both in vitro before transplantation and in vivo after transplantation. Despite one fourth dosage of MSCs, significantly more PKH26-labeled donor cells were recovered with intratracheal administration than with intraperitoneal administration both during normoxia and hyperoxia. The hyperoxia-induced increase in the number of TUNEL-positive cells, myeloperoixdase activity, and the level of IL-6 mRNA were significantly attenuated with both intratracheal and intraperitoneal MSCs transplantation. However, the hyperoxia-induced impaired alveolarization and increased the level of TNF-alpha and TGF-beta mRNA, alpha-SMA protein, and collagen were significantly attenuated only with intratracheal MSCs transplantation. MSCs differentiated into respiratory epithelium in vitro and a few PKH26-positive donor cells were colocalized with pro surfactant protein C in the damaged lungs. In conclusion, intratracheal transplantation of human UCB-derived MSCs is more effective than intraperitoneal transplantation in attenuating the hyperoxia-induced lung injury in neonatal rats.

Regional delivery of mesothelin-targeted CAR T cell therapy generates potent and long-lasting CD4-dependent tumor immunity

PubMed Central

Adusumilli, Prasad S.; Cherkassky, Leonid; Villena-Vargas, Jonathan; Colovos, Christos; Servais, Elliot; Plotkin, Jason; Jones, David R.; Sadelain, Michel

2015-01-01

Translating the recent success of chimeric antigen receptor (CAR) T cell therapy for hematological malignancies to solid tumors will necessitate overcoming several obstacles, including inefficient T cell tumor infiltration and insufficient functional persistence. Taking advantage of an orthotopic model that faithfully mimics human pleural malignancy, we evaluated two routes of administration of mesothelin-targeted T cells using the M28z CAR. We found that intra-pleurally administered CAR T cells vastly out-performed systemically infused T cells, requiring 30-fold fewer M28z T cells to induce long-term complete remissions. Following intrapleural T cell administration, prompt in vivo antigen-induced T cell activation allowed robust CAR T cell expansion and effector differentiation, resulting in enhanced anti-tumor efficacy and functional T cell persistence for 200 days. Regional T cell administration also promoted efficient elimination of extrathoracic tumor sites. This therapeutic efficacy was dependent on early CD4+ T cell activation associated with a higher intra-tumoral CD4/CD8 cell ratios and CD28-dependent CD4+ T cell-mediated cytotoxicity. In contrast, intravenously delivered CAR T cells, even when accumulated at equivalent numbers in the pleural tumor, did not achieve comparable activation, tumor eradication or persistence. The remarkable ability of intrapleurally administered T cells to circulate and persist supports the concept of delivering optimal CAR T cell therapy through “regional distribution centers.” Based on these results, we are opening a phase I clinical trial to evaluate the safety of intrapleural administration of mesothelin-targeted CAR T cells in patients with primary or secondary pleural malignancies. PMID:25378643

Human umbilical cord blood mesenchymal stem cells reduce colitis in mice by activating NOD2 signaling to COX2.

PubMed

Kim, Hyung-Sik; Shin, Tae-Hoon; Lee, Byung-Chul; Yu, Kyung-Rok; Seo, Yoojin; Lee, Seunghee; Seo, Min-Soo; Hong, In-Sun; Choi, Soon Won; Seo, Kwang-Won; Núñez, Gabriel; Park, Jong-Hwan; Kang, Kyung-Sun

2013-12-01

Decreased levels or function of nucleotide-binding oligomerization domain 2 (NOD2) are associated with Crohn's disease. NOD2 regulates intestinal inflammation, and also is expressed by human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs), to regulate their differentiation. We investigated whether NOD2 is required for the anti-inflammatory activities of MSCs in mice with colitis. Colitis was induced in mice by administration of dextran sulfate sodium or trinitrobenzene sulfonic acid. Mice then were given intraperitoneal injections of NOD2-activated hUCB-MSCs; colon tissues and mesenteric lymph nodes were collected for histologic analyses. A bromodeoxyuridine assay was used to determine the ability of hUCB-MSCs to inhibit proliferation of human mononuclear cells in culture. Administration of hUCB-MSCs reduced the severity of colitis in mice. The anti-inflammatory effects of hUCB-MSCs were greatly increased by activation of NOD2 by its ligand, muramyl dipeptide (MDP). Administration of NOD2-activated hUCB-MSCs increased anti-inflammatory responses in colons of mice, such as production of interleukin (IL)-10 and infiltration by T regulatory cells, and reduced production of inflammatory cytokines. Proliferation of mononuclear cells was inhibited significantly by co-culture with hUCB-MSCs that had been stimulated with MDP. MDP induced prolonged production of prostaglandin (PG)E2 in hUCB-MSCs via the NOD2-RIP2 pathway, which suppressed proliferation of mononuclear cells derived from hUCB. PGE2 produced by hUCB-MSCs in response to MDP increased production of IL-10 and T regulatory cells. In mice, production of PGE2 by MSCs and subsequent production of IL-10 were required to reduce the severity of colitis. Activation of NOD2 is required for the ability of hUCB-MSCs to reduce the severity of colitis in mice. NOD2 signaling increases the ability of these cells to suppress mononuclear cell proliferation by inducing production of PGE2. Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.

Posttransplant Intramuscular Injection of PLX-R18 Mesenchymal-Like Adherent Stromal Cells Improves Human Hematopoietic Engraftment in A Murine Transplant Model

PubMed Central

Metheny, Leland; Eid, Saada; Lingas, Karen; Ofir, Racheli; Pinzur, Lena; Meyerson, Howard; Lazarus, Hillard M.; Huang, Alex Y.

2018-01-01

Late-term complications of hematopoietic cell transplantation (HCT) are numerous and include incomplete engraftment. One possible mechanism of incomplete engraftment after HCT is cytokine-mediated suppression or dysfunction of the bone marrow microenvironment. Mesenchymal stromal cells (MSCs) elaborate cytokines that nurture or stimulate the marrow microenvironment by several mechanisms. We hypothesize that the administration of exogenous MSCs may modulate the bone marrow milieu and improve peripheral blood count recovery in the setting of incomplete engraftment. In the current study, we demonstrated that posttransplant intramuscular administration of human placental derived mesenchymal-like adherent stromal cells [PLacental eXpanded (PLX)-R18] harvested from a three-dimensional in vitro culture system improved posttransplant engraftment of human immune compartment in an immune-deficient murine transplantation model. As measured by the percentage of CD45+ cell recovery, we observed improvement in the peripheral blood counts at weeks 6 (8.4 vs. 24.1%, p < 0.001) and 8 (7.3 vs. 13.1%, p < 0.05) and in the bone marrow at week 8 (28 vs. 40.0%, p < 0.01) in the PLX-R18 cohort. As measured by percentage of CD19+ cell recovery, there was improvement at weeks 6 (12.6 vs. 3.8%) and 8 (10.1 vs. 4.1%). These results suggest that PLX-R18 may have a therapeutic role in improving incomplete engraftment after HCT. PMID:29520362

Posttransplant Intramuscular Injection of PLX-R18 Mesenchymal-Like Adherent Stromal Cells Improves Human Hematopoietic Engraftment in A Murine Transplant Model.

PubMed

Metheny, Leland; Eid, Saada; Lingas, Karen; Ofir, Racheli; Pinzur, Lena; Meyerson, Howard; Lazarus, Hillard M; Huang, Alex Y

2018-01-01

Late-term complications of hematopoietic cell transplantation (HCT) are numerous and include incomplete engraftment. One possible mechanism of incomplete engraftment after HCT is cytokine-mediated suppression or dysfunction of the bone marrow microenvironment. Mesenchymal stromal cells (MSCs) elaborate cytokines that nurture or stimulate the marrow microenvironment by several mechanisms. We hypothesize that the administration of exogenous MSCs may modulate the bone marrow milieu and improve peripheral blood count recovery in the setting of incomplete engraftment. In the current study, we demonstrated that posttransplant intramuscular administration of human placental derived mesenchymal-like adherent stromal cells [PLacental eXpanded (PLX)-R18] harvested from a three-dimensional in vitro culture system improved posttransplant engraftment of human immune compartment in an immune-deficient murine transplantation model. As measured by the percentage of CD45 + cell recovery, we observed improvement in the peripheral blood counts at weeks 6 (8.4 vs. 24.1%, p  < 0.001) and 8 (7.3 vs. 13.1%, p  < 0.05) and in the bone marrow at week 8 (28 vs. 40.0%, p  < 0.01) in the PLX-R18 cohort. As measured by percentage of CD19 + cell recovery, there was improvement at weeks 6 (12.6 vs. 3.8%) and 8 (10.1 vs. 4.1%). These results suggest that PLX-R18 may have a therapeutic role in improving incomplete engraftment after HCT.

Acridine Orange/exosomes increase the delivery and the effectiveness of Acridine Orange in human melanoma cells: A new prototype for theranostics of tumors.

PubMed

Iessi, Elisabetta; Logozzi, Mariantonia; Lugini, Luana; Azzarito, Tommaso; Federici, Cristina; Spugnini, Enrico Pierluigi; Mizzoni, Davide; Di Raimo, Rossella; Angelini, Daniela F; Battistini, Luca; Cecchetti, Serena; Fais, Stefano

2017-12-01

Specifically targeted drug delivery systems with low immunogenicity and toxicity are deemed to increase efficacy of cancer chemotherapy. Acridine Orange (AO) is an acidophilic dye with a strong tumoricidal action following excitation with a light source at 466 nm. However, to date the clinical use of AO is limited by the potential side effects elicited by systemic administration. The endogenous nanocarrier exosomes have been recently introduced as a natural delivery system for therapeutic molecules. In this article, we show the outcome of the administration to human melanoma cells of AO charged Exosomes (Exo-AO), in both monolayer and spheroid models. The results showed an extended drug delivery time of Exo-AO to melanoma cells as compared to the free AO, improving the cytotoxicity of AO. This study shows that Exo-AO have a great potential for a real exploitation as a new theranostic approach against tumors based on AO delivered through the exosomes.

A novel imidazopyridine analogue as a phosphatidylinositol 3-kinase inhibitor against human breast cancer.

PubMed

Lee, Hyunseung; Li, Guang-Yong; Jeong, Yujeong; Jung, Kyung Hee; Lee, Ju-Hee; Ham, Kyungrok; Hong, Sungwoo; Hong, Soon-Sun

2012-05-01

Potentiation of anti-breast cancer activity of an imidazopyridine-based PI3Kα inhibitor, HS-104, was investigated in human breast cancer cells. HS-104 shows strong inhibitory activity against recombinant PI3Kα isoform and the PI3K signaling pathway, resulting in anti-proliferative activity in breast cancer cells. It also induced cell cycle arrest at the G(2)/M phase as well as apoptosis. Furthermore, oral administration of HS-104 significantly inhibited the growth of tumor in SkBr3 mouse xenograft models. Therefore, HS-104 could be considered as a potential candidate for the treatment of human breast cancer. Crown Copyright © 2011. Published by Elsevier Ireland Ltd. All rights reserved.

Autoreactive T Cells and Chronic Fungal Infection Drive Esophageal Carcinogenesis

PubMed Central

Zhu, Feng; Willette-Brown, Jami; Song, Na-Young; Lomada, Dakshayani; Song, Yongmei; Xue, Liyan; Gray, Zane; Zhao, Zitong; Davis, Sean R.; Sun, Zhonghe; Zhang, Peilin; Wu, Xiaolin; Zhan, Qimin; Richie, Ellen R.; Hu, Yinling

2018-01-01

SUMMARY Humans with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a T cell–driven autoimmune disease caused by impaired central tolerance, are susceptible to developing chronic fungal infection and esophageal squamous cell carcinoma (ESCC). However, the relationship between autoreactive T cells and chronic fungal infection in ESCC development remains unclear. We find that kinase-dead Ikkα knockin mice develop phenotypes reminiscent of APECED, including impaired central tolerance, autoreactive T cells, chronic fungal infection, and ESCCs expressing specific human ESCC markers. Using this model, we investigated the potential link between ESCC and fungal infection. Autoreactive CD4 T cells permit fungal infection and incite tissue injury and inflammation. Antifungal treatment or depletion of autoreactive CD4 T cells rescues, whereas oral fungal administration promotes, ESCC development. Inhibition of inflammation or EGFR activity decreases fungal burden. Importantly, fungal infection is highly associated with ESCCs in non-autoimmune human patients. Therefore, autoreactive T cells and chronic fungal infection, fostered by inflammation and epithelial injury, promote ESCC development. PMID:28407484

Pathology of the testicle and sex accessory glands following the administration of boldenone and boldione as growth promoters in veal calves.

PubMed

Cannizzo, Francesca T; Zancanaro, Gabriele; Spada, Francesca; Mulasso, Chiara; Biolatti, Bartolomeo

2007-11-01

Boldenone and its precursor Boldione are illegally used for anabolic purposes in humans, horses and cattle. To develop more effective policies and programs to maximize food security, Italian Public Health Services investigate all indicators capable of assisting the recognition of treated animals, and prioritize research and the formulation of action strategies for the promotion of healthy eating. Thus, an experimental administration of boldenone and boldione at anabolic dosages in veal calves was carried out to evaluate the changes in target organs by qualitative and semi-quantitative morphological analysis. The lesions resembled the effects already observed after the administration of androgen hormones to cattle. Main findings were represented by prostate hypersecretion, increased rate of apoptotic cells and decreased rate of Ki67 positive cells in the germ cell line of treated animals, particularly in boldione group and finally some new features like hypertrophy of the prostate urothelial cells.

Antibacterial Effect of Human Mesenchymal Stem Cells Is Mediated in Part from Secretion of the Antimicrobial Peptide LL-37

PubMed Central

Krasnodembskaya, Anna; Song, Yuanlin; Fang, Xiaohui; Gupta, Naveen; Serikov, Vladimir; Lee, Jae-Woo; Matthay, Michael A.

2012-01-01

Recent in vivo studies indicate that mesenchymal stem cells (MSCs) may have beneficial effects in the treatment of sepsis induced by bacterial infection. Administration of MSCs in these studies improved survival and enhanced bacterial clearance. The primary objective of this study was to test the hypothesis that human MSCs possessed intrinsic antimicrobial properties. We studied the effect of human MSCs derived from bone marrow on the bacterial growth of Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria. MSCs as well as their conditioned medium (CM) demonstrated marked inhibition of bacterial growth in comparison with control medium or normal human lung fibroblasts (NHLF). Analysis of expression of major antimicrobial peptides indicated that one of the factors responsible for the antimicrobial activity of MSC CM against Gram-negative bacteria was the human cathelicidin antimicrobial peptide, hCAP-18/LL-37. Both m-RNA and protein expression data showed that the expression of LL-37 in MSCs increased after bacterial challenge. Using an in vivo mouse model of E. coli pneumonia, intratracheal administration of MSCs reduced bacterial growth (in colony-forming unit) in the lung homogenates and in the bronchoalveolar lavage (BAL) fluid, and administration of MSCs simultaneously with a neutralizing antibody to LL-37 resulted in a decrease in bacterial clearance. In addition, the BAL itself from MSC-treated mice had a greater antimicrobial activity in comparison with the BAL of phosphate buffered saline (PBS)-treated mice. Human bone marrow-derived MSCs possess direct antimicrobial activity, which is mediated in part by the secretion of human cathelicidin hCAP-18/LL-37. PMID:20945332

Induction of Proteinases in the Human Preovulatory Follicle of the Menstrual Cycle by hCG

PubMed Central

Rosewell, Katherine L.; Al-Alem, Linah; Zakerkish, Farnosh; McCord, Lauren; Akin, James W.; Chaffin, Charles L.; Brännström, Mats; Curry, Thomas E.

2014-01-01

Objective To explore the temporal expression in granulosa and theca cells of key members of the MMP and ADAMTS families across the periovulatory period in women in order to gain insight into their possible roles during ovulation and early luteinization. Design Experimental prospective clinical study and laboratory-based investigation. Setting University Medical Center and private IVF center. Animal and Patient(s) Thirty eight premenopausal women undergoing surgery for tubal ligation and 6 premenopausal women undergoing ART. Intervention(s) Administration of hCG and harvesting of follicles by laparoscopy and collection of granulosa-lutein cells at oocyte retrieval. Main Outcome Measure(s) Expression of mRNA for MMPs and ADAMTSs in human granulosa cells and theca cells collected across the periovulatory period of the menstrual cycle and in cultured granulosa-lutein cells after hCG. Localization of MMPs and ADAMTSs by immunohistochemistry. Result(s) Expression of MMP1 and MMP19 mRNA increased in both granulosa and theca cells after hCG administration. ADAMTS1 and ADAMTS 9 mRNA increased in granulosa cells after hCG treatment, however thecal cell expression for ADAMTS1 was unchanged while ADAMTS9 expression was decreased. Expression of MMP8 and MMP13 mRNA was unchanged. Immunohistochemistry confirmed the localization of MMP1, MMP19, ADAMTS1 and ADAMTS9 to the granulosa and thecal cell layers. Conclusion(s) The collection of the dominant follicle throughout the periovulatory period has allowed the identification of proteolytic remodeling enzymes in the granulosa and theca compartments that may be critically involved in human ovulation. These proteinases may work in concert to regulate breakdown of the follicular wall and release of the oocyte. PMID:25516084

Bilirubin Inhibits Neointima Formation and Vascular Smooth Muscle Cell Proliferation and Migration

PubMed Central

Peyton, Kelly J.; Shebib, Ahmad R.; Azam, Mohammad A.; Liu, Xiao-ming; Tulis, David A.; Durante, William

2012-01-01

Bilirubin is a heme metabolite generated by the concerted action of the enzymes heme oxygenase and biliverdin reductase. Although long considered a toxic byproduct of heme catabolism, recent preclinical, and clinical studies indicate the bilirubin exerts beneficial effects in the circulation. In the present study, we determined whether local administration of bilirubin attenuates neointima formation following injury of rat carotid arteries. In addition, the ability of bilirubin to regulate the proliferation and migration of human arterial smooth muscle cells (SMCs) was investigated. Local perivascular administration of bilirubin immediately following balloon injury of rat carotid arteries significantly attenuated neointima formation. Bilirubin-mediated inhibition of neointimal thickening was associated with a significant decrease in ERK activity and cyclin D1 and A protein expression, and an increase in p21 and p53 protein expression in injured blood vessels. Treatment of human aortic SMCs with bilirubin inhibited proliferation and migration in a concentration-dependent manner without affecting cell viability. In addition, bilirubin resulted in a concentration-dependent increase in the percentage of cells in the G0/G1 phase of the cell cycle and this was paralleled by a decrease in the fraction of cells in the S and G2M phases of the cell cycle. Finally, bilirubin had no effect on mitochondrial function and ATP content of vascular SMCs. In conclusion, these studies demonstrate that bilirubin inhibits neointima formation after arterial injury and this is associated with alterations in the expression of cell cycle regulatory proteins. Furthermore, bilirubin blocks proliferation and migration of human arterial SMCs and arrests SMCs in the G0/G1 phase of the cell cycle. Bilirubin represents an attractive therapeutic agent in treating occlusive vascular disease. PMID:22470341

A549 Cells: Lung Carcinoma Cell Line for Adenovirus | NCI Technology Transfer Center | TTC

Cancer.gov

Scientists at the National Cancer Institute have developed a cell line designated A549 that was derived from explanted cultures of human lung cancer tissue. The A549 cell line has been tested under the guidance of the United States Food and Drug Administration (FDA) so, under current Good Manufacturing Practices (GMP), these cells may be suitable for use in manufacturing constructs for use in clinical trials. The National Cancer Institute seeks parties to non-exclusively license this research material.

Antitumor and antiangiogenic effects of GA-13315, a gibberellin derivative.

PubMed

Zhang, Yanli; Zhang, Hui; Chen, Jingbo; Zhao, Haixia; Zeng, Xianghui; Zhang, Hongbin; Qing, Chen

2012-02-01

This study showed that 13-chlorine-3,15-dioxy-gibberellic acid methyl ester (GA-13315), a gibberellin derivative, possessed high antitumor and antiangiogenic activity in vitro and in vivo. Cytotoxicity assays showed that GA-13315 was a potential and efficient antitumor compound, with inhibitory concentration 50 (IC(50)) values ranging from 0.13 to 30.28 μg/ml in 12 human tumor cell lines, and it showed moderate toxicity to peripheral blood mononuclear cells with an IC(50) value of 14.2 μg/ml. Administration of 0.5 or 2.5 mg/kg GA-13315 for 23 days significantly inhibited tumor growth of human non-small cell lung tumor (A549) xenografts, with relative growth rates ranging from 29.91% to 35.05%. Acute toxicity was determined in ICR mice, and the lethal dose 50 (LD(50)) was 4.19 g/kg after intragastric administration. The high antitumor potency of GA-13315 occurred in parallel with its antiangiogenic activity. In vitro, GA-13315 inhibited recombinant human epithelial growth factor-induced chemotactic motility and capillary-like tube formation of primary cultured human endothelial cells. Furthermore, GA-13315 decreased the factor VIII(+) microvessel density and vascular endothelial growth factor expression in A549 tumors, indicating its antiangiogenic efficacy in vivo. These results indicate that the antiangiogenic activity of GA-13315 contributes to its anticancer properties. Further studies are needed to investigate the use of GA-13315 as an anticancer drug.

Technique, pharmacokinetics, toxicity, and efficacy of intratumoral etanidazole and radiotherapy for treatment of spontaneous feline oral squamous cell carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Evans, S.M.; LaCreta, F.; Helfand, S.

1991-04-01

The histologic appearance, locoregional recurrence, and rate/site of metastases of spontaneous feline oral squamous cell carcinoma are similar to head and neck cancer in humans. A feasibility study of intratumoral Etanidazole, a hypoxic cell sensitizer, and radiation therapy were instituted in this model. Eleven cats with feline squamous cell carcinoma were treated with intratumoral Etanidazole and radiation therapy. Total Etanidazole doses were 1.5-24.0 gms/m2 (0.5-6.9 gms). The tumor partial response rate was 100% (11/11); the median volume regression was 70%. All cats have died as a result of tumor recurrence or tumor-related complications. Median survival was 116 days. Ten catsmore » have been autopsied. Non-necrotic and necrotic tumor cells were identified at the treatment site in all cats. Pharmacokinetic studies were performed in six cats. Following intravenous infusion, the plasma elimination of the Etanidazole was biexponential. The systemic availability following intratumoral administration was 61.2 +/- 21.1%. Peak plasma Etanidazole levels were observed 14 minutes following intratumoral injection, after which elimination was biexponential. Thirty minutes following intratumoral Etanidazole administration, tumor Etanidazole levels were 62.8% of plasma levels. Feline squamous cell carcinoma appears to be a useful model of human head and neck cancer. Cats tolerate substantial doses of intratumoral and intravenous Etanidazole. Etanidazole and radiation therapy cause rapid regression, but not cure, of feline squamous cell carcinoma. There is a similarity between the intravenous kinetics of Etanidazole in humans and cats. Further studies in this model are planned.« less

Histamine release and fibrinogen adsorption mediate acute inflammatory responses to biomaterial implants in humans

PubMed Central

Zdolsek, Johann; Eaton, John W; Tang, Liping

2007-01-01

Background Medical implants often fail as a result of so-called foreign body reactions during which inflammatory cells are recruited to implant surfaces. Despite the clinical importance of this phenomenon, the mechanisms involved in these reactions to biomedical implants in humans are not well understood. The results from animal studies suggest that both fibrinogen adsorption to the implant surface and histamine release by local mast cells are involved in biomaterial-mediated acute inflammatory responses. The purpose of this study was to test this hypothesis in humans. Methods Thirteen male medical student volunteers (Caucasian, 21–30 years of age) were employed for this study. To assess the importance of fibrinogen adsorption, six volunteers were implanted with polyethylene teraphthalate disks pre-coated with their own (fibrinogen-containing) plasma or (fibrinogen-free) serum. To evaluate the importance of histamine, seven volunteers were implanted with uncoated disks with or without prior oral administration of histamine receptor antagonists. The acute inflammatory response was estimated 24 hours later by measuring the activities of implant-associated phagocyte-specific enzymes. Results Plasma coated implants accumulated significantly more phagocytes than did serum coated implants and the recruited cells were predominantly macrophage/monocytes. Administration of both H1 and H2 histamine receptor antagonists greatly reduced the recruitment of macrophages/monocytes and neutrophils on implant surfaces. Conclusion In humans – as in rodents – biomaterial-mediated inflammatory responses involve at least two crucial events: histamine-mediated phagocyte recruitment and phagocyte accumulation on implant surfaces engendered by spontaneously adsorbed host fibrinogen. Based on these results, we conclude that reducing fibrinogen:surface interactions should enhance biocompatibility and that administration of histamine receptor antagonists prior to, and shortly after, medical device implantation should improve the functionality and longevity of medical implants. PMID:17603911

Nitric oxide-induced changes in endothelial expression of phosphodiesterases 2, 3, and 5.

PubMed

Schankin, Christoph J; Kruse, Lars S; Reinisch, Veronika M; Jungmann, Steffen; Kristensen, Julie C; Grau, Stefan; Ferrari, Uta; Sinicina, Inga; Goldbrunner, Roland; Straube, Andreas; Kruuse, Christina

2010-03-01

To investigate nitric oxide (NO)-mediated changes in expression of cyclic nucleotide degrading phosphodiesterases 2A (PDE2A), PDE3B, and PDE5A in human endothelial cells. Nitric oxide induces production of cyclic guanosine monophosphate (cGMP), which along with cyclic adenosine monophosphate (cAMP) is degraded by PDEs. NO donors and selective inhibitors of PDE3 and PDE5 induce migraine-like headache and play a role in endothelial dysfunction during stroke. The current study investigates possible NO modulation of cGMP-related PDEs relevant to headache induction in a cell line containing such PDEs. Real time polymerase chain reaction and Western blots were used to show expression of PDE2A, PDE3B, and PDE5A in a stable cell line of human brain microvascular endothelial cells. Effects of NO on PDE expression were analyzed at specific time intervals after continued DETA NONOate administration. This study shows the expression of PDE2A, PDE3B, and PDE5A mRNA and PDE3B and PDE5A protein in human cerebral endothelial cells. Long-term DETA NONOate administration induced an immediate mRNA up-regulation of PDE5A (1.9-fold, 0.5 hour), an early peak of PDE2A (1.4-fold, 1 and 2 hours) and later up-regulation of both PDE3B (1.6-fold, 4 hours) and PDE2A (1.7-fold, 8 hours and 1.2-fold after 24 hours). Such changes were, however, not translated into significant changes in protein expression indicating few, if any, functional effects. Long-term NO stimulation modulated PDE3 and PDE5 mRNA expression in endothelial cells. However, PDE3 and PDE5 protein levels were unaffected by NO. The presence of PDE3 or PDE5 in endothelial cells indicates that selective inhibitors may have functional effects in such cells. A complex interaction of cGMP and cAMP in response to NO administration may take place if the mRNA translates into active protein. Whether or not this plays a role in the headache mechanisms remains to be investigated.

Immunotherapy of metastatic melanoma by reversal of immune suppression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Biggs, M.W.; Eiselein, J.E.

1997-01-01

Beginning with the observation that the human enteorvirus, Poliovirus Sabin 1, will lyse human melanoma cells in culture, clinical trials involving two patients with advance melanoma were performed. Parenteral injection of the viable Poliovirus into cutaneous melanoma metastases followed in 24 hours by oral administration of cyclophosphamide. The results of these two trials are described.

[Efficacy of HSV-tk/GCV system on human laryngeal squamous cell cancer in vitro].

PubMed

Ding, Xiu-yong; Qin, Yong; Li, Fu-ying; Cong, Tie-chuan

2006-05-01

Efficacy of HSV-tk/GCV system antitumor effects was assessed on human laryngeal cancer cell line Hep-2 in vitro. To assess the HSV-tk/CGV system whether has an antitumour effect on human laryngeal squamous cell cancer Hep-2 in vitro. The mechanisms of cytotoxity were also assessed. Hep-2 cells were transfected with HSV-tk gene by lipofection. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the HSV-tk gene expression. MTT was utilized to test for the cytotoxicity of this system. The cell-circle arrest and apoptosis were analyzed by flowcytometry assay. HSV-tk gene transfected cells demonstrated obvious cytoreductivity followed by ganciclovir (GCV) administration and this cytoreductivity showed partial GCV dose-independent. HSV-tk gene transfected cells demonstrated obvious s-phase arrest, no apoptosis and necrosis occurred. The HSV-tk/GCV system can inhabit the growth of Hep-2 cells effectively. S-phase arrest perhaps is the main reason that leads to the cell inhibition in our study. HSV-tk/GCV system has potential antitumor effects for the future clinical practice.

Intraperitoneal administration of tumor-targeting Salmonella typhimurium A1-R inhibits disseminated human ovarian cancer and extends survival in nude mice

PubMed Central

Zhang, Yong; Zhao, Ming; Yano, Shuya; Uehara, Fuminari; Yamamoto, Mako; Hiroshima, Yukihiko; Toneri, Makoto; Bouvet, Michael; Matsubara, Hisahiro; Tsuchiya, Hiroyuki; Hoffman, Robert M.

2015-01-01

Peritoneal disseminated cancer is highly treatment resistant. We here report the efficacy of intraperitoneal (i.p.) administration of tumor-targeting Salmonella typhimurium A1-R in a nude mouse model of disseminated human ovarian cancer. The mouse model was established by intraperitoneal injection of the human ovarian cancer cell line SKOV3-GFP. Seven days after implantation, mice were treated with S. typhimurium A1-R via intravenous (i.v.) or i.p. administration at the same dose, 5×107 CFU, once per week. Both i.v. and i.p. treatments effected prolonged survival compared with the untreated control group (P=0.025 and P<0.001, respectively). However, i.p. treatment was less toxic than i.v. treatment. Tumor-specific targeting of S. typhimurium A1-R was confirmed with bacterial culture from tumors and various organs and tumor or organ colony formation after i.v. or i.p. injection. Selective tumor targeting was most effective with i.p. administration. The results of the present study show S. typhimurium A1-R has promising clinical potential for disseminated ovarian cancer, especially via i.p. administration. PMID:25957417

Human mesenchymal stromal cell transplantation modulates neuroinflammatory milieu in a mouse model of amyotrophic lateral sclerosis.

PubMed

Boido, Marina; Piras, Antonio; Valsecchi, Valeria; Spigolon, Giada; Mareschi, Katia; Ferrero, Ivana; Vizzini, Andrea; Temi, Santa; Mazzini, Letizia; Fagioli, Franca; Vercelli, Alessandro

2014-08-01

Mesenchymal stromal cells (MSCs), after intraparenchymal, intrathecal and endovenous administration, have been previously tested for cell therapy in amyotrophic lateral sclerosis in the SOD1 (superoxide dismutase 1) mouse. However, every administration route has specific pros and cons. We administrated human MSCs (hMSCs) in the cisterna lumbaris, which is easily accessible and could be used in outpatient surgery, in the SOD1 G93A mouse, at the earliest onset of symptoms. Control animals received saline injections. Motor behavior was checked starting from 2 months of age until the mice were killed. Animals were killed 2 weeks after transplantation; lumbar motoneurons were stereologically counted, astrocytes and microglia were analyzed and quantified after immunohistochemistry and cytokine expression was assayed by means of real-time polymerase chain reaction. We provide evidence that this route of administration can exert strongly positive effects. Motoneuron death and motor decay were delayed, astrogliosis was reduced and microglial activation was modulated. In addition, hMSC transplantation prevented the downregulation of the anti-inflammatory interleukin-10, as well as that of vascular endothelial growth factor observed in saline-treated transgenic mice compared with wild type, and resulted in a dramatic increase in the expression of the anti-inflammatory interleukin-13. Our results suggest that hMSCs, when intracisternally administered, can exert their paracrine potential, influencing the inflammatory response of the host. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Targeting PI3K-AKT-mTOR by LY3023414 inhibits human skin squamous cell carcinoma cell growth in vitro and in vivo.

PubMed

Zou, Ying; Ge, Minggai; Wang, Xuemin

2017-08-19

Abnormal activation of PI3K-AKT-mTOR signaling is detected in human skin squamous cell carcinoma (SCC). LY3023414 is a novel, potent, and orally bio-available PI3K-AKT-mTOR inhibitor. Its activity against human skin SCC cells was tested. We demonstrated that LY3023414 was cytotoxic when added to established (A431 line) and primary (patient-derived) human skin SCC cells. LY3023414 induced G0/1-S arrest and inhibited proliferation of skin SCC cells. Moreover, LY3023414 induced activation of caspase-3/-9 and apoptosis in skin SCC cells. Intriguingly, LY3023414 was yet non-cytotoxic nor pro-apoptotic to normal human skin cells (melanocytes, keratinocytes and fibroblasts). At the molecular level, LY3023414 blocked PI3K-AKT-mTOR activation in skin SCC cells, as it dephosphorylated PI3K-AKT-mTOR substrates: P85, AKT and S6K1. In vivo studies showed that oral administration of LY3023414 at well-tolerated doses inhibited A431 xenograft tumor growth in severe combined immunodeficiency (SCID) mice. AKT-mTOR activation in LY3023414-treated tumors was also largely inhibited. Together, these results suggest that targeting PI3K-AKT-mTOR by LY3023414 inhibits human skin SCC cell growth in vitro and in vivo, establishing the rationale for further clinical testing. Copyright © 2017 Elsevier Inc. All rights reserved.

Efficient generation of megakaryocytes from human induced pluripotent stem cells using food and drug administration-approved pharmacological reagents.

PubMed

Liu, Yanfeng; Wang, Ying; Gao, Yongxing; Forbes, Jessica A; Qayyum, Rehan; Becker, Lewis; Cheng, Linzhao; Wang, Zack Z

2015-04-01

Megakaryocytes (MKs) are rare hematopoietic cells in the adult bone marrow and produce platelets that are critical to vascular hemostasis and wound healing. Ex vivo generation of MKs from human induced pluripotent stem cells (hiPSCs) provides a renewable cell source of platelets for treating thrombocytopenic patients and allows a better understanding of MK/platelet biology. The key requirements in this approach include developing a robust and consistent method to produce functional progeny cells, such as MKs from hiPSCs, and minimizing the risk and variation from the animal-derived products in cell cultures. In this study, we developed an efficient system to generate MKs from hiPSCs under a feeder-free and xeno-free condition, in which all animal-derived products were eliminated. Several crucial reagents were evaluated and replaced with Food and Drug Administration-approved pharmacological reagents, including romiplostim (Nplate, a thrombopoietin analog), oprelvekin (recombinant interleukin-11), and Plasbumin (human albumin). We used this method to induce MK generation from hiPSCs derived from 23 individuals in two steps: generation of CD34(+)CD45(+) hematopoietic progenitor cells (HPCs) for 14 days; and generation and expansion of CD41(+)CD42a(+) MKs from HPCs for an additional 5 days. After 19 days, we observed abundant CD41(+)CD42a(+) MKs that also expressed the MK markers CD42b and CD61 and displayed polyploidy (≥16% of derived cells with DNA contents >4N). Transcriptome analysis by RNA sequencing revealed that megakaryocytic-related genes were highly expressed. Additional maturation and investigation of hiPSC-derived MKs should provide insights into MK biology and lead to the generation of large numbers of platelets ex vivo. ©AlphaMed Press.

Repeated hematopoietic stem and progenitor cell mobilization without depletion of the bone marrow stem and progenitor cell pool in mice after repeated administration of recombinant murine G-CSF.

PubMed

de Kruijf, Evert-Jan F M; van Pel, Melissa; Hagoort, Henny; Kruysdijk, Donnée; Molineux, Graham; Willemze, Roel; Fibbe, Willem E

2007-05-01

Administration of recombinant-human G-CSF (rhG-CSF) is highly efficient in mobilizing hematopoietic stem and progenitor cells (HSC/HPC) from the bone marrow (BM) toward the peripheral blood. This study was designed to investigate whether repeated G-CSF-induced HSC/HPC mobilization in mice could lead to a depletion of the bone marrow HSC/HPC pool with subsequent loss of mobilizing capacity. To test this hypothesis Balb/c mice were treated with a maximum of 12 repeated 5-day cycles of either 10 microg rhG-CSF/day or 0.25 microg rmG-CSF/day. Repeated administration of rhG-CSF lead to strong inhibition of HSC/HPC mobilization toward the peripheral blood and spleen after >4 cycles because of the induction of anti-rhG-CSF antibodies. In contrast, after repeated administration of rmG-CSF, HSC/HPC mobilizing capacity remained intact for up to 12 cycles. The number of CFU-GM per femur did not significantly change for up to 12 cycles. We conclude that repeated administration of G-CSF does not lead to depletion of the bone marrow HSC/HPC pool.

Chemical carcinogenesis: Too many rodent carcinogens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ames, B.N.; Gold, L.S.

1990-10-01

The administration of chemicals at the maximum tolerated dose (MTD) in standard animal cancer tests is postulated to increase cell division (mitogenesis), which in turn increases rates of mutagenesis and thus carcinogenesis. The animal data are consistent with this mechanism, because a high proportion{endash}about half{endash}of all chemicals tested (whether natural or synthetic) are indeed rodent carcinogens. The authors conclude that at the low doses of most human exposures, where cell killing does not occur, the hazards to humans of rodent carcinogens may be much lower than is commonly assumed.

In situ induction of dendritic cell–based T cell tolerance in humanized mice and nonhuman primates

PubMed Central

Jung, Kyeong Cheon; Jeon, Yoon Kyung; Ban, Young Larn; Min, Hye Sook; Kim, Eun Ji; Kim, Ju Hyun; Kang, Byung Hyun; Bae, Youngmee; Yoon, Il-Hee; Kim, Yong-Hee; Lee, Jae-Il; Kim, Jung-Sik; Shin, Jun-Seop; Yang, Jaeseok; Kim, Sung Joo; Rostlund, Emily; Muller, William A.

2011-01-01

Induction of antigen-specific T cell tolerance would aid treatment of diverse immunological disorders and help prevent allograft rejection and graft versus host disease. In this study, we establish a method of inducing antigen-specific T cell tolerance in situ in diabetic humanized mice and Rhesus monkeys receiving porcine islet xenografts. Antigen-specific T cell tolerance is induced by administration of an antibody ligating a particular epitope on ICAM-1 (intercellular adhesion molecule 1). Antibody-mediated ligation of ICAM-1 on dendritic cells (DCs) led to the arrest of DCs in a semimature stage in vitro and in vivo. Ablation of DCs from mice completely abrogated anti–ICAM-1–induced antigen-specific T cell tolerance. T cell responses to unrelated antigens remained unaffected. In situ induction of DC-mediated T cell tolerance using this method may represent a potent therapeutic tool for preventing graft rejection. PMID:22025302

AHR Activation Is Protective against Colitis Driven by T Cells in Humanized Mice.

PubMed

Goettel, Jeremy A; Gandhi, Roopali; Kenison, Jessica E; Yeste, Ada; Murugaiyan, Gopal; Sambanthamoorthy, Sharmila; Griffith, Alexandra E; Patel, Bonny; Shouval, Dror S; Weiner, Howard L; Snapper, Scott B; Quintana, Francisco J

2016-10-25

Existing therapies for inflammatory bowel disease that are based on broad suppression of inflammation result in variable clinical benefit and unwanted side effects. A potential therapeutic approach for promoting immune tolerance is the in vivo induction of regulatory T cells (Tregs). Here we report that activation of the aryl hydrocarbon receptor using the non-toxic agonist 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) induces human Tregs in vitro that suppress effector T cells through a mechanism mediated by CD39 and Granzyme B. We then developed a humanized murine system whereby human CD4 + T cells drive colitis upon exposure to 2,4,6-trinitrobenzenesulfonic acid and assessed ITE as a potential therapeutic. ITE administration ameliorated colitis in humanized mice with increased CD39, Granzyme B, and IL10-secreting human Tregs. These results develop an experimental model to investigate human CD4 + T responses in vivo and identify the non-toxic AHR agonist ITE as a potential therapy for promoting immune tolerance in the intestine. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Stabilizing short-lived Schiff base derivatives of 5-aminouracils that activate mucosal-associated invariant T cells

NASA Astrophysics Data System (ADS)

Mak, Jeffrey Y. W.; Xu, Weijun; Reid, Robert C.; Corbett, Alexandra J.; Meehan, Bronwyn S.; Wang, Huimeng; Chen, Zhenjun; Rossjohn, Jamie; McCluskey, James; Liu, Ligong; Fairlie, David P.

2017-03-01

Mucosal-associated invariant T (MAIT) cells are activated by unstable antigens formed by reactions of 5-amino-6-D-ribitylaminouracil (a vitamin B2 biosynthetic intermediate) with glycolysis metabolites such as methylglyoxal. Here we show superior preparations of antigens in dimethylsulfoxide, avoiding their rapid decomposition in water (t1/2 1.5 h, 37 °C). Antigen solution structures, MAIT cell activation potencies (EC50 3-500 pM), and chemical stabilities are described. Computer analyses of antigen structures reveal stereochemical and energetic influences on MAIT cell activation, enabling design of a water stable synthetic antigen (EC50 2 nM). Like native antigens, this antigen preparation induces MR1 refolding and upregulates surface expression of human MR1, forms MR1 tetramers that detect MAIT cells in human PBMCs, and stimulates cytokine expression (IFNγ, TNF) by human MAIT cells. These antigens also induce MAIT cell accumulation in mouse lungs after administration with a co-stimulant. These chemical and immunological findings provide new insights into antigen properties and MAIT cell activation.

Method of increasing radiation sensitivity by inhibition of beta one integrin

DOEpatents

Park, Catherine [San Francisco, CA; Bissell, Mina J [Berkeley, CA

2009-11-17

A method for increasing or monitoring apoptosis in tumor cells by the co-administration of ionizing radiation and an anti-integrin antibody. Increasing apoptosis reduces tumor growth in vivo and in a cell culture model. The antibody is directed against the beta-1 integrin subunit and is inhibitory of beta-1 integrin signaling. Other molecules having an inhibitory effect on beta-1 integrin, either in signaling or in binding to its cognate extracellular receptors may also be used. The present method is particularly of interest in treatment of tumor cells associated with breast cancer, wherein radiation is currently used alone. The present method further contemplates a monoclonal antibody suitable for human administration that may further comprise a radioisotope attached thereto.

Therapeutic effect of mesenchymal multipotent stromal cells on memory in animals with Alzheimer-type neurodegeneration.

PubMed

Bobkova, N V; Poltavtseva, R A; Samokhin, A N; Sukhikh, G T

2013-11-01

Transplantation of human mesenchymal multipotent stromal cells improved spatial memory in bulbectomized mice with Alzheimer-type neurodegeneration. The positive effect was observed in 1 month after intracerebral transplantation and in 3 months after systemic injection of mesenchymal multipotent stromal cells. No cases of malignant transformation were noted. These findings indicate prospects of using mesenchymal multipotent stromal cells for the therapy of Alzheimer disease and the possibility of their systemic administration for attaining the therapeutic effect.

Lactoferrin for prevention of common viral infections.

PubMed

Wakabayashi, Hiroyuki; Oda, Hirotsugu; Yamauchi, Koji; Abe, Fumiaki

2014-11-01

Although lactoferrin has many biological functions, the host-protective effects against pathogenic microorganisms including bacteria, fungi, and viruses are regarded as one of the most important. Here, we review research on the protective role of lactoferrin administration against common viral infections. Many studies have shown the in vitro antiviral activity of lactoferrin against viral pathogens that cause common infections such as the common cold, influenza, gastroenteritis, summer cold, and herpes, where lactoferrin inhibits mainly viral attachment to the target cells. Recently, studies indicating the in vivo protective effects of lactoferrin by oral administration against common viral infections have been increasing. For instance, norovirus is an extremely important emerging human pathogen that causes a majority of gastroenteritis outbreaks worldwide that may be a target candidate for lactoferrin. Lactoferrin consumption reduced the incidence of noroviral gastroenteritis in children and a similar effect was observed in a wide range of ages in a preliminary survey. A recent in vitro study reported that lactoferrin inhibits both cellular attachment of the murine norovirus, a virus closely-related to the human norovirus, and viral replication in the cells by inducing antiviral cytokines interferon (IFN)-α/β. Lactoferrin administration also enhances NK cell activity and Th1 cytokine responses, which lead to protection against viral infections. In conclusion, lactoferrin consumption may protect the host from viral infections through inhibiting the attachment of a virus to the cells, replication of the virus in the cells, and enhancement of systemic immune functions. Copyright © 2014 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Recombinant HSP70 and mild heat shock stimulate growth of aged mesenchymal stem cells.

PubMed

Andreeva, N V; Zatsepina, O G; Garbuz, D G; Evgen'ev, M B; Belyavsky, A V

2016-07-01

Heat shock proteins including the major stress protein HSP70 support intracellular homeostasis and prevent protein damage after a temperature increase and other stressful environmental stimuli, as well as during aging. We have shown earlier that prolonged administration of recombinant human HSP70 to mice exhibiting Alzheimer's-like neurodegeneration as well as during sepsis reduces the clinical manifestations of these pathologies. Herein, we studied the action of recombinant human HSP70 on young and aged mouse mesenchymal stem cells (MSCs) in culture. The results obtained indicate that HSP70 at concentrations of 2 μg/ml and higher significantly stimulates growth of aged but not young MSCs. A similar effect is produced by application of a mild heat shock (42 °C 5 min) to the cells. Importantly, responses of young and aged MSCs to heat shock treatment of various durations differed drastically, and aged MSCs were significantly more sensitive to higher heat stress exposures than the young cells. Western blotting and protein labeling experiments demonstrated that neither mild heat shock nor exogenous HSP70 administration resulted in significant endogenous HSP70 induction in young and aged MSCs, whereas mild heat shock increased HSC70 levels in aged MSCs. The results of this study suggest that the administration of exogenous HSP70 and the application of mild heat stress may produce a certain "rejuvenating" effect on MSCs and possibly other cell types in vivo, and these interventions may potentially be used for life extension by delaying various manifestations of aging at the molecular and cellular level.

The anti-hepatocellular carcinoma cell activity by a novel mTOR kinase inhibitor CZ415

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Wei; Research Center of Blood Transfusion Medicine, Education Ministry Key Laboratory of Laboratory Medicine, Zhejiang Provincial People's Hospital, People’s Hospital of Hangzhou medical College, Hangzhou; Chen, Bingyu

Dysregulation of mammalian target of rapamycin (mTOR) in hepatocellular carcinoma (HCC) represents a valuable treatment target. Recent studies have developed a highly-selective and potent mTOR kinase inhibitor, CZ415. Here, we showed that nM concentrations of CZ415 efficiently inhibited survival and induced apoptosis in HCC cell lines (HepG2 and Huh-7) and primary-cultured human HCC cells. Meanwhile, CZ415 inhibited proliferation of HCC cells, more potently than mTORC1 inhibitors (rapamycin and RAD001). CZ415 was yet non-cytotoxic to the L02 human hepatocytes. Mechanistic studies showed that CZ415 disrupted assembly of mTOR complex 1 (mTORC1) and mTORC2 in HepG2 cells. Meanwhile, activation of mTORC1 (p-S6K1)more » and mTORC2 (p-AKT, Ser-473) was almost blocked by CZ415. In vivo studies revealed that oral administration of CZ415 significantly suppressed HepG2 xenograft tumor growth in severe combined immuno-deficient (SCID) mice. Activation of mTORC1/2 was also largely inhibited in CZ415-treated HepG2 tumor tissue. Together, these results show that CZ415 blocks mTORC1/2 activation and efficiently inhibits HCC cell growth in vitro and in vivo. - Highlights: • CZ415 is anti-survival and pro-apoptotic to hepatocellular carcinoma (HCC) cells. • CZ415 inhibits HCC cell proliferation, more efficiently than mTORC1 inhibitors. • CZ415 blocks assembly and activation of both mTORC1 and mTORC2 in HCC cells. • CZ415 oral administration inhibits HepG2 tumor growth in SCID mice. • mTORC1/2 activation in HepG2 tumor is inhibited with CZ415 administration.« less

From isolation to implantation: a concise review of mesenchymal stem cell therapy in bone fracture repair

PubMed Central

2014-01-01

Compromised bone-regenerating capability following a long bone fracture is often the result of reduced host bone marrow (BM) progenitor cell numbers and efficacy. Without surgical intervention, these malunions result in mobility restrictions, deformities, and disability. The clinical application of BM-derived mesenchymal stem cells (MSCs) is a feasible, minimally invasive therapeutic option to treat non-union fractures. This review focuses on novel, newly identified cell surface markers in both the mouse and human enabling the isolation and purification of osteogenic progenitor cells as well as their direct and indirect contributions to fracture repair upon administration. Furthermore, clinical success to date is summarized with commentary on autologous versus allogeneic cell sources and the methodology of cell administration. Given our clinical success to date in combination with recent advances in the identification, isolation, and mechanism of action of MSCs, there is a significant opportunity to develop improved technologies for defining therapeutic MSCs and potential to critically inform future clinical strategies for MSC-based bone regeneration. PMID:25099622

Current perspectives on protective roles of erythropoietin in cardiovascular system: erythropoietin receptor as a novel therapeutic target.

PubMed

Kagaya, Yutaka; Asaumi, Yasuhide; Wang, Wanting; Takeda, Morihiko; Nakano, Makoto; Satoh, Kimio; Fukumoto, Yoshihiro; Shimokawa, Hiroaki

2012-06-01

Erythropoietin (EPO) is a principal regulator that promotes proliferation and terminal differentiation of erythroid progenitor cells. EPO receptors are expressed not only in hematopoietic lineage cells but also in the cardiovascular system. We performed animal experiments using transgene-rescued EPO receptor null mutant mice (EpoR-/- rescued) that express the EPO receptor exclusively in the hematopoietic cells. The results of these experiments suggest that endogenous EPO/EPO receptor system in the heart exerts cardioprotective effects against myocardial injury induced by ischemia followed by reperfusion and pressure-overload induced left ventricular dysfunction. Many animal experiments have shown that the administration of recombinant human EPO also elicits cardioprotective effects against myocardial injury induced by ischemia and reperfusion. In contrast to the promising results of these animal experiments, recent clinical trials failed to demonstrate the reduction in infarct size or improvement of cardiac function by the administration of recombinant human EPO in patients with acute myocardial infarction who underwent primary percutaneous coronary intervention. It should be tested in future clinical studies whether a relatively low dose of recombinant human EPO or its derivatives that have no erythropoietic action reduces infarct size and ameliorates cardiac dysfunction in patients with acute myocardial infarction. In this article, we review implications of anemia associated with chronic heart failure, roles of the endogenous EPO/EPO receptor system, and the effects of the administration of erythropoiesis-stimulating agents in pathologic conditions of the heart by focusing on the EPO receptor as a potential candidate of novel therapeutic targets in cardiovascular diseases.

Human Neutrophil Peptides Mediate Endothelial-Monocyte Interaction, Foam Cell Formation, and Platelet Activation

PubMed Central

Quinn, Kieran L.; Henriques, Melanie; Tabuchi, Arata; Han, Bing; Yang, Hong; Cheng, Wei-Erh; Tole, Soumitra; Yu, Hanpo; Luo, Alice; Charbonney, Emmanuel; Tullis, Elizabeth; Lazarus, Alan; Robinson, Lisa A.; Ni, Heyu; Peterson, Blake R.; Kuebler, Wolfgang M.; Slutsky, Arthur S.; Zhang, Haibo

2016-01-01

Objective Neutrophils are involved in the inflammatory responses during atherosclerosis. Human neutrophil peptides (HNPs) released from activated neutrophils exert immune modulating properties. We hypothesized that HNPs play an important role in neutrophil-mediated inflammatory cardiovascular responses in atherosclerosis. Methods and Results We examined the role of HNPs in endothelial-leukocyte interaction, platelet activation, and foam cell formation in vitro and in vivo. We demonstrated that stimulation of human coronary artery endothelial cells with clinically relevant concentrations of HNPs resulted in monocyte adhesion and transmigration; induction of oxidative stress in human macrophages, which accelerates foam cell formation; and activation and aggregation of human platelets. The administration of superoxide dismutase or anti-CD36 antibody reduced foam cell formation and cholesterol efflux. Mice deficient in double genes of low-density lipoprotein receptor and low-density lipoprotein receptor–related protein (LRP), and mice deficient in a single gene of LRP8, the only LRP phenotype expressed in platelets, showed reduced leukocyte rolling and decreased platelet aggregation and thrombus formation in response to HNP stimulation. Conclusion HNPs exert proatherosclerotic properties that appear to be mediated through LRP8 signaling pathways, suggesting an important role for HNPs in the development of inflammatory cardiovascular diseases. PMID:21817096

Investigation of the cytotoxicity, apoptosis and pharmacokinetics of Raddeanin A.

PubMed

Gu, Guiying; Qi, Huanhuan; Jiang, Tianyue; Ma, Bo; Fang, Zheng; Xu, Hong; Zhang, Qi

2017-03-01

Raddeanin A, one of the triterpenoid saponins extracted from Anemone raddeana rhizome of the Ranunculaceae family, has demonstrated the ability to inhibit the growth of human hepatic and gastric cancer cells. However, the effects of Raddeanin A on human colon cancer cells have not been investigated extensively. The present study aimed to examine the antiproliferative and apoptosis-inducing effects of Raddeanin A on the HCT-116 human colon cancer cell line in vitro , and evaluate the pharmacokinetic and biodistribution properties of Raddeanin A in mice following a single oral administration. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to assess the in vitro cytotoxicity of Raddeanin A against HCT-116 cells. 4',6-Diamidino-2-phenylindole, dihydrochloride staining and flow cytometry were performed to further examine the apoptosis-inducing capability of Raddeanin A. The concentrations of Raddeanin A in the plasma and tissues were analyzed using liquid chromatography-tandem mass spectrometry. Raddeanin A showed a dose-dependent antiproliferative effect towards the HCT-116 cells, with a half maximal inhibitory concentration of ~1.4 µM. Treatment with Raddeanin A resulted in a significant induction of apoptosis, observed as apparent morphological changes of the nuclei, with a total apoptotic ratio of 41.8% at a concentration of 3 µM. Low concentrations of Raddeanin A were detected in the heart, liver, spleen, lung, kidney and plasma of the mice following oral administration, however, the majority of the Raddeanin A was distributed in the intestinal tract, particularly in the colon and caecum. These present study confirmed the growth-inhibitory and apoptosis-inducing effects of Raddeanin A on HCT-116 cells and performed preliminary examinations of its pharmacokinetic properties, which provide a foundation for further investigating the inhibitory mechanism on the colon cancer cells in vivo .

PolyI:C and mouse survivin artificially embedding human 2B peptide induce a CD4+ T cell response to autologous survivin in HLA-A*2402 transgenic mice.

PubMed

Kasamatsu, Jun; Takahashi, Shojiro; Azuma, Masahiro; Matsumoto, Misako; Morii-Sakai, Akiko; Imamura, Masahiro; Teshima, Takanori; Takahashi, Akari; Hirohashi, Yoshihiko; Torigoe, Toshihiko; Sato, Noriyuki; Seya, Tsukasa

2015-01-01

CD4(+) T cell effectors are crucial for establishing antitumor immunity. Dendritic cell maturation by immune adjuvants appears to facilitate subset-specific CD4(+) T cell proliferation, but the adjuvant effect for CD4 T on induction of cytotoxic T lymphocytes (CTLs) is largely unknown. Self-antigenic determinants with low avidity are usually CD4 epitopes in mutated proteins with tumor-associated class I-antigens (TAAs). In this study, we made a chimeric version of survivin, a target of human CTLs. The chimeric survivin, where human survivin-2B containing a TAA was embedded in the mouse survivin frame (MmSVN2B), was used to immunize HLA-A-2402/K(b)-transgenic (HLA24(b)-Tg) mice. Subcutaneous administration of MmSVN2B or xenogeneic human survivin (control HsSNV2B) to HLA24(b)-Tg mice failed to induce an immune response without co-administration of an RNA adjuvant polyI:C, which was required for effector induction in vivo. Although HLA-A-2402/K(b) presented the survivin-2B peptide in C57BL/6 mice, 2B-specific tetramer assays showed that no CD8(+) T CTLs specific to survivin-2B proliferated above the detection limit in immunized mice, even with polyI:C treatment. However, the CD4(+) T cell response, as monitored by IFN-γ, was significantly increased in mice given polyI:C+MmSVN2B. The Th1 response and antibody production were enhanced in the mice with polyI:C. The CD4 epitope responsible for effector function was not Hs/MmSNV13-27, a nonconserved region between human and mouse survivin, but region 53-67, which was identical between human and mouse survivin. These results suggest that activated, self-reactive CD4(+) helper T cells proliferate in MmSVN2B+polyI:C immunization and contribute to Th1 polarization followed by antibody production, but hardly participate in CTL induction. Copyright © 2014 Elsevier GmbH. All rights reserved.

Intratracheal Administration of Recombinant Human Keratinocyte Growth Factor Promotes Alveolar Epithelial Cell Proliferation during Compensatory Lung Growth in Rat

PubMed Central

Furukawa, Katsuro; Matsumoto, Keitaro; Nagayasu, Takeshi; Yamamoto-Fukuda, Tomomi; Tobinaga, Shuichi; Abo, Takafumi; Yamasaki, Naoya; Tsuchiya, Tomoshi; Miyazaki, Takuro; Kamohara, Ryotaro; Nanashima, Atsushi; Obatake, Masayuki; Koji, Takehiko

2013-01-01

Keratinocyte growth factor (KGF) is considered to be one of the most important mitogens for lung epithelial cells. The objectives of this study were to confirm the effectiveness of intratracheal injection of recombinant human KGF (rhKGF) during compensatory lung growth and to optimize the instillation protocol. Here, trilobectomy in adult rat was performed, followed by intratracheal rhKGF instillation with low (0.4 mg/kg) and high (4 mg/kg) doses at various time-points. The proliferation of alveolar cells was assessed by the immunostaining for proliferating cell nuclear antigen (PCNA) in the residual lung. We also investigated other immunohistochemical parameters such as KGF, KGF receptor and surfactant protein A as well as terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Consequently, intratracheal single injection of rhKGF in high dose group significantly increased PCNA labeling index (LI) of alveolar cells in the remaining lung. Surprisingly, there was no difference in PCNA LI between low and high doses of rhKGF with daily injection, and PCNA LI reached a plateau level with 2 days-consecutive administration (about 60%). Our results indicate that even at low dose, daily intratracheal injection is effective to maintain high proliferative states during the early phase of compensatory lung growth. PMID:24610965

Intratracheal Administration of Recombinant Human Keratinocyte Growth Factor Promotes Alveolar Epithelial Cell Proliferation during Compensatory Lung Growth in Rat.

PubMed

Furukawa, Katsuro; Matsumoto, Keitaro; Nagayasu, Takeshi; Yamamoto-Fukuda, Tomomi; Tobinaga, Shuichi; Abo, Takafumi; Yamasaki, Naoya; Tsuchiya, Tomoshi; Miyazaki, Takuro; Kamohara, Ryotaro; Nanashima, Atsushi; Obatake, Masayuki; Koji, Takehiko

2013-12-28

Keratinocyte growth factor (KGF) is considered to be one of the most important mitogens for lung epithelial cells. The objectives of this study were to confirm the effectiveness of intratracheal injection of recombinant human KGF (rhKGF) during compensatory lung growth and to optimize the instillation protocol. Here, trilobectomy in adult rat was performed, followed by intratracheal rhKGF instillation with low (0.4 mg/kg) and high (4 mg/kg) doses at various time-points. The proliferation of alveolar cells was assessed by the immunostaining for proliferating cell nuclear antigen (PCNA) in the residual lung. We also investigated other immunohistochemical parameters such as KGF, KGF receptor and surfactant protein A as well as terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Consequently, intratracheal single injection of rhKGF in high dose group significantly increased PCNA labeling index (LI) of alveolar cells in the remaining lung. Surprisingly, there was no difference in PCNA LI between low and high doses of rhKGF with daily injection, and PCNA LI reached a plateau level with 2 days-consecutive administration (about 60%). Our results indicate that even at low dose, daily intratracheal injection is effective to maintain high proliferative states during the early phase of compensatory lung growth.

Human mesenchymal stem cells suppress donor CD4(+) T cell proliferation and reduce pathology in a humanized mouse model of acute graft-versus-host disease.

PubMed

Tobin, L M; Healy, M E; English, K; Mahon, B P

2013-05-01

Acute graft-versus-host disease (aGVHD) is a life-threatening complication following allogeneic haematopoietic stem cell transplantation (HSCT), occurring in up to 30-50% of patients who receive human leucocyte antigen (HLA)-matched sibling transplants. Current therapies for steroid refractory aGVHD are limited, with the prognosis of patients suboptimal. Mesenchymal stem or stromal cells (MSC), a heterogeneous cell population present in many tissues, display potent immunomodulatory abilities. Autologous and allogeneic ex-vivo expanded human MSC have been utilized to treat aGVHD with promising results, but the mechanisms of therapeutic action remain unclear. Here a robust humanized mouse model of aGVHD based on delivery of human peripheral blood mononuclear cells (PBMC) to non-obese diabetic (NOD)-severe combined immunodeficient (SCID) interleukin (IL)-2rγ(null) (NSG) mice was developed that allowed the exploration of the role of MSC in cell therapy. MSC therapy resulted in the reduction of liver and gut pathology and significantly increased survival. Protection was dependent upon the timing of MSC therapy, with conventional MSC proving effective only after delayed administration. In contrast, interferon (IFN)-γ-stimulated MSC were effective when delivered with PBMC. The beneficial effect of MSC therapy in this model was not due to the inhibition of donor PBMC chimerism, as CD45(+) and T cells engrafted successfully in this model. MSC therapy did not induce donor T cell anergy, FoxP3(+) T regulatory cells or cause PBMC apoptosis in this model; however, it was associated with the direct inhibition of donor CD4(+) T cell proliferation and reduction of human tumour necrosis factor-α in serum. © 2012 British Society for Immunology.

Phthalocyanine-Peptide Conjugates for Epidermal Growth Factor Receptor Targeting1

PubMed Central

Ongarora, Benson G.; Fontenot, Krystal R.; Hu, Xiaoke; Sehgal, Inder; Satyanarayana-Jois, Seetharama D.; Vicente, M. Graça H.

2012-01-01

Four phthalocyanine (Pc)-peptide conjugates designed to target the epidermal growth factor receptor (EGFR) were synthesized and evaluated in vitro using four cell lines: human carcinoma A431 and HEp2, human colorectal HT-29, and kidney Vero (negative control) cells. Two peptide ligands for EGFR were investigated: EGFR-L1 and -L2, bearing 6 and 13 amino acid residues, respectively. The peptides and Pc-conjugates were shown to bind to EGFR using both theoretical (Autodock) and experimental (SPR) investigations. The Pc-EGFR-L1 conjugates 5a and 5b efficiently targeted EGFR and were internalized, in part due to their cationic charge, whereas the uncharged Pc-EGFR-L2 conjugates 4b and 6a poorly targeted EGFR maybe due to their low aqueous solubility. All conjugates were non-toxic (IC50 > 100 µM) to HT-29 cells, both in the dark and upon light activation (1 J/cm2). Intravenous (iv) administration of conjugate 5b into nude mice bearing A431 and HT-29 human tumor xenografts resulted in a near-IR fluorescence signal at ca. 700 nm, 24 h after administration. Our studies show that Pc-EGFR-L1 conjugates are promising near-IR fluorescent contrast agents for CRC, and potentially other EGFR over-expressing cancers. PMID:22468711

Lipid-core nanocapsules as a nanomedicine for parenteral administration of tretinoin: development and in vitro antitumor activity on human myeloid leukaemia cells.

PubMed

Ourique, A F; Azoubel, S; Ferreira, C V; Silva, C B; Marchiori, M C L; Pohlmann, A R; Guterres, S S; Beck, R C R

2010-06-01

Tretinoin-loaded conventional nanocapsules have showed a significant protection of this drug against UVC radiation. However, this formulation presents a limited stability on storage. We hypothesized that the association of tretinoin to lipid-core nanocapsules could increase the physicochemical stability of such formulations, focusing on the development of a reliable nanomedicine for parenteral administration. However, this advantage should still be accompanied by the known photoprotective effect of conventional polymeric nanocapsules against the exposure of tretinoin to UV radiation. Results showed that tretinoin-loaded lipid-core nanocapsules improved the physicochemical stability of formulations under storage, without changing their ability to protect tretinoin either against UVA or UVC radiation. In addition, the effect of nanoencapsulation on the antiproliferative and differentiation properties of tretinoin was studied on human myeloid leukemia cells (HL60 cells) showing that tretinoin-loaded lipid-core nanocapsules presents a longer antitumor efficiency compared to the free tretinoin. These results allow us to propose the current formulation (tretinoin-loaded lipid-core nanocapsules) as a promising parenteral nanomedicine for the treatment of acute promyelocytic leukaemia.

Optimal Route for Human Umbilical Cord Blood-Derived Mesenchymal Stem Cell Transplantation to Protect Against Neonatal Hyperoxic Lung Injury: Gene Expression Profiles and Histopathology.

PubMed

Sung, Dong Kyung; Chang, Yun Sil; Ahn, So Yoon; Sung, Se In; Yoo, Hye Soo; Choi, Soo Jin; Kim, Soo Yoon; Park, Won Soon

2015-01-01

The aim of this study was to determine the optimal route of mesenchymal stem cell (MSC) transplantation. To this end, gene expression profiling was performed to compare the effects of intratracheal (i.t.) versus intravenous (i.v.) MSC administration. Furthermore, the therapeutic efficacy of each route to protect against neonatal hyperoxic lung injury was also determined. Newborn Sprague-Dawley rats were exposed to hyperoxia (90% oxygen) from birth for 14 days. Human umbilical cord blood-derived MSCs labeling with PKH26 were transplanted through either the i.t. (5×10(5)) or i.v. (2×10(6)) route at postnatal day (P) 5. At P14, lungs were harvested for histological, biochemical and microarray analyses. Hyperoxic conditions induced an increase in the mean linear intercept and mean alveolar volume (MAV), indicative of impaired alveolarization. The number of ED-1 positive cells was significantly decreased by both i.t. and i.v. transplantations. However, i.t. administration of MSCs resulted in a greater decrease in MAV and ED-1 positive cells compared to i.v. administration. Moreover, the number of TUNEL-positive cells was significantly decreased in the i.t. group, but not in the i.v. group. Although the i.t. group received only one fourth of the number of MSCs that the i.v. group did, a significantly higher number of donor cell-derived red PKH 26 positivity were recovered in the i.t. group. Hyperoxic conditions induced the up regulation of genes associated with the inflammatory response, such as macrophage inflammatory protein-1 α, tumor necrosis factor-α and inter leukin-6; genes associated with cell death, such as p53 and caspases; and genes associated with fibrosis, such as connective tissue growth factor. In contrast, hyperoxic conditions induced the dwon-regulation of vascular endothelial growth factor and hepatocyte growth factor. These hyperoxia-induced changes in gene expression were decreased in the i.t. group, but not in the i.v. group. Thus, local i.t. MSC transplantation was more effective than systemic i.v. MSC administration in protecting against neonatal hyperoxic lung injury.

Optimal Route for Human Umbilical Cord Blood-Derived Mesenchymal Stem Cell Transplantation to Protect Against Neonatal Hyperoxic Lung Injury: Gene Expression Profiles and Histopathology

PubMed Central

Ahn, So Yoon; Sung, Se In; Yoo, Hye Soo; Choi, Soo Jin; Kim, Soo Yoon; Park, Won Soon

2015-01-01

The aim of this study was to determine the optimal route of mesenchymal stem cell (MSC) transplantation. To this end, gene expression profiling was performed to compare the effects of intratracheal (IT) versus intravenous (IV) MSC administration. Furthermore, the therapeutic efficacy of each route to protect against neonatal hyperoxic lung injury was also determined. Newborn Sprague-Dawley rats were exposed to hyperoxia (90% oxygen) from birth for 14 days. Human umbilical cord blood-derived MSCs labeling with PKH26 were transplanted through either the IT (5×105) or IV (2×106) route at postnatal day (P) 5. At P14, lungs were harvested for histological, biochemical and microarray analyses. Hyperoxic conditions induced an increase in the mean linear intercept and mean alveolar volume (MAV), indicative of impaired alveolarization. The number of ED-1 positive cells was significantly decreased by both IT and IV transplantations. However, IT administration of MSCs resulted in a greater decrease in MAV and ED-1 positive cells compared to IV administration. Moreover, the number of TUNEL-positive cells was significantly decreased in the IT group, but not in the IV group. Although the IT group received only one fourth of the number of MSCs that the IV group did, a significantly higher number of donor cell-derived red PKH 26 positivity were recovered in the IT group. Hyperoxic conditions induced the up regulation of genes associated with the inflammatory response, such as macrophage inflammatory protein-1 α, tumor necrosis factor-α and inter leukin-6; genes associated with cell death, such as p53 and caspases; and genes associated with fibrosis, such as connective tissue growth factor. In contrast, hyperoxic conditions induced the dwon-regulation of vascular endothelial growth factor and hepatocyte growth factor. These hyperoxia-induced changes in gene expression were decreased in the IT group, but not in the IV group. Thus, local IT MSC transplantation was more effective than systemic IV MSC administration in protecting against neonatal hyperoxic lung injury. PMID:26305093

Identification of cells initiating human melanomas.

PubMed

Schatton, Tobias; Murphy, George F; Frank, Natasha Y; Yamaura, Kazuhiro; Waaga-Gasser, Ana Maria; Gasser, Martin; Zhan, Qian; Jordan, Stefan; Duncan, Lyn M; Weishaupt, Carsten; Fuhlbrigge, Robert C; Kupper, Thomas S; Sayegh, Mohamed H; Frank, Markus H

2008-01-17

Tumour-initiating cells capable of self-renewal and differentiation, which are responsible for tumour growth, have been identified in human haematological malignancies and solid cancers. If such minority populations are associated with tumour progression in human patients, specific targeting of tumour-initiating cells could be a strategy to eradicate cancers currently resistant to systemic therapy. Here we identify a subpopulation enriched for human malignant-melanoma-initiating cells (MMIC) defined by expression of the chemoresistance mediator ABCB5 (refs 7, 8) and show that specific targeting of this tumorigenic minority population inhibits tumour growth. ABCB5+ tumour cells detected in human melanoma patients show a primitive molecular phenotype and correlate with clinical melanoma progression. In serial human-to-mouse xenotransplantation experiments, ABCB5+ melanoma cells possess greater tumorigenic capacity than ABCB5- bulk populations and re-establish clinical tumour heterogeneity. In vivo genetic lineage tracking demonstrates a specific capacity of ABCB5+ subpopulations for self-renewal and differentiation, because ABCB5+ cancer cells generate both ABCB5+ and ABCB5- progeny, whereas ABCB5- tumour populations give rise, at lower rates, exclusively to ABCB5- cells. In an initial proof-of-principle analysis, designed to test the hypothesis that MMIC are also required for growth of established tumours, systemic administration of a monoclonal antibody directed at ABCB5, shown to be capable of inducing antibody-dependent cell-mediated cytotoxicity in ABCB5+ MMIC, exerted tumour-inhibitory effects. Identification of tumour-initiating cells with enhanced abundance in more advanced disease but susceptibility to specific targeting through a defining chemoresistance determinant has important implications for cancer therapy.

Preclinical evaluation of the efficacy, pharmacokinetics and immunogenicity of JS-001, a programmed cell death protein-1 (PD-1) monoclonal antibody

PubMed Central

Fu, Jie; Wang, Fang; Dong, Li-hou; Zhang, Jing; Deng, Cheng-lian; Wang, Xue-li; Xie, Xin-yao; Zhang, Jing; Deng, Ruo-xian; Zhang, Li-bo; Wu, Hai; Feng, Hui; Chen, Bo; Song, Hai-feng

2017-01-01

JS-001 is the first monoclonal antibody (mAb) against programmed cell death protein-1 (PD-1) approved by the China Food and Drug Administration (CFDA) into the clinical trails. To date, however, no pre-clinical pharmacological and pharmacokinetic (PK) data are available. In this study, we investigated the efficacy of JS-001 and conducted a preclinical PK study, including the monitoring of anti-drug antibodies (ADAs). We found that JS-001 specifically bound to PD-1 antigen with an EC50 of 21 nmol/L, and competently blocked the binding of PD-1 antigen to PD-L1 and PD-L2 with IC50 of 3.0 and 3.1 nmol/L, respectively. Furthermore, JS-001 displayed distinct species cross-reactivity: it could bind to the PD-1 antigen on the peripheral blood mononuclear cells (PBMCs) of humans and cynomolgus monkeys, but not to those of mice and woodchucks; the Kd values for the interaction between JS-001 and PD-1 antigens on CD8+ T cells of human and cynomolgus monkey were 2.1 nmol/L and 1.2 nmol/L, respectively. In vitro, treatment with JS-001 (0.01–10 μg/mL) dose-dependently stimulated human T cell proliferation, as well as IFN-γ and TNF-α secretion. In HBsAg-vaccinated cynomolgus monkeys, the expression of PD-1+/CD4+ and PD-1+/CD8+ was significantly elevated, intramuscular injection of JS-001 (1 and 10 mg/kg) resulted in dramatic decreases in PD-1+/CD4+ and PD-1+/CD8+ expression in a dose-dependent manner, which was supported by PD-1 receptor occupancy (RO) results. In the PK study, 18 cynomolgus monkeys treated with single, ascending doses of 1, 10, and 75 mg/kg, and another 6 cynomolgus monkeys received 10 mg/kg successive administration. The plasma clearance of JS-001 followed a linear PK profile with single administration in the 1 and 10 mg/kg groups and a non-linear PK profile in the 75 mg/kg group. In the successive 10 mg/kg administration group, no drug accumulation was observed. But the AUC from the last exposure was lower than that of the first administration, which was probably due to the production of ADAs, as demonstrated in immunogenicity study. These non-clinical data are encouraging and provide a basis for the efficacy and safety of JS-001 in clinical trials. PMID:28317872

Preclinical evaluation of the efficacy, pharmacokinetics and immunogenicity of JS-001, a programmed cell death protein-1 (PD-1) monoclonal antibody.

PubMed

Fu, Jie; Wang, Fang; Dong, Li-Hou; Zhang, Jing; Deng, Cheng-Lian; Wang, Xue-Li; Xie, Xin-Yao; Zhang, Jing; Deng, Ruo-Xian; Zhang, Li-Bo; Wu, Hai; Feng, Hui; Chen, Bo; Song, Hai-Feng

2017-05-01

JS-001 is the first monoclonal antibody (mAb) against programmed cell death protein-1 (PD-1) approved by the China Food and Drug Administration (CFDA) into the clinical trails. To date, however, no pre-clinical pharmacological and pharmacokinetic (PK) data are available. In this study, we investigated the efficacy of JS-001 and conducted a preclinical PK study, including the monitoring of anti-drug antibodies (ADAs). We found that JS-001 specifically bound to PD-1 antigen with an EC 50 of 21 nmol/L, and competently blocked the binding of PD-1 antigen to PD-L1 and PD-L2 with IC 50 of 3.0 and 3.1 nmol/L, respectively. Furthermore, JS-001 displayed distinct species cross-reactivity: it could bind to the PD-1 antigen on the peripheral blood mononuclear cells (PBMCs) of humans and cynomolgus monkeys, but not to those of mice and woodchucks; the K d values for the interaction between JS-001 and PD-1 antigens on CD8 + T cells of human and cynomolgus monkey were 2.1 nmol/L and 1.2 nmol/L, respectively. In vitro, treatment with JS-001 (0.01-10 μg/mL) dose-dependently stimulated human T cell proliferation, as well as IFN-γ and TNF-α secretion. In HBsAg-vaccinated cynomolgus monkeys, the expression of PD-1 + /CD4 + and PD-1 + /CD8 + was significantly elevated, intramuscular injection of JS-001 (1 and 10 mg/kg) resulted in dramatic decreases in PD-1 + /CD4 + and PD-1 + /CD8 + expression in a dose-dependent manner, which was supported by PD-1 receptor occupancy (RO) results. In the PK study, 18 cynomolgus monkeys treated with single, ascending doses of 1, 10, and 75 mg/kg, and another 6 cynomolgus monkeys received 10 mg/kg successive administration. The plasma clearance of JS-001 followed a linear PK profile with single administration in the 1 and 10 mg/kg groups and a non-linear PK profile in the 75 mg/kg group. In the successive 10 mg/kg administration group, no drug accumulation was observed. But the AUC from the last exposure was lower than that of the first administration, which was probably due to the production of ADAs, as demonstrated in immunogenicity study. These non-clinical data are encouraging and provide a basis for the efficacy and safety of JS-001 in clinical trials.

Flow Cytometric Analysis of Mononuclear Phagocytes in Nondiseased Human Lung and Lung-Draining Lymph Nodes.

PubMed

Desch, A Nicole; Gibbings, Sophie L; Goyal, Rajni; Kolde, Raivo; Bednarek, Joe; Bruno, Tullia; Slansky, Jill E; Jacobelli, Jordan; Mason, Robert; Ito, Yoko; Messier, Elise; Randolph, Gwendalyn J; Prabagar, Miglena; Atif, Shaikh M; Segura, Elodie; Xavier, Ramnik J; Bratton, Donna L; Janssen, William J; Henson, Peter M; Jakubzick, Claudia V

2016-03-15

The pulmonary mononuclear phagocyte system is a critical host defense mechanism composed of macrophages, monocytes, monocyte-derived cells, and dendritic cells. However, our current characterization of these cells is limited because it is derived largely from animal studies and analysis of human mononuclear phagocytes from blood and small tissue resections around tumors. Phenotypic and morphologic characterization of mononuclear phagocytes that potentially access inhaled antigens in human lungs. We acquired and analyzed pulmonary mononuclear phagocytes from fully intact nondiseased human lungs (including the major blood vessels and draining lymph nodes) obtained en bloc from 72 individual donors. Differential labeling of hematopoietic cells via intrabronchial and intravenous administration of antibodies within the same lobe was used to identify extravascular tissue-resident mononuclear phagocytes and exclude cells within the vascular lumen. Multiparameter flow cytometry was used to identify mononuclear phagocyte populations among cells labeled by each route of antibody delivery. We performed a phenotypic analysis of pulmonary mononuclear phagocytes isolated from whole nondiseased human lungs and lung-draining lymph nodes. Five pulmonary mononuclear phagocytes were observed, including macrophages, monocyte-derived cells, and dendritic cells that were phenotypically distinct from cell populations found in blood. Different mononuclear phagocytes, particularly dendritic cells, were labeled by intravascular and intrabronchial antibody delivery, countering the notion that tissue and blood mononuclear phagocytes are equivalent systems. Phenotypic descriptions of the mononuclear phagocytes in nondiseased lungs provide a precedent for comparative studies in diseased lungs and potential targets for therapeutics.

Impact of irradiation and immunosuppressive agents on immune system homeostasis in rhesus macaques

PubMed Central

Meyer, C; Walker, J; Dewane, J; Engelmann, F; Laub, W; Pillai, S; Thomas, Charles R; Messaoudi, I

2015-01-01

In this study we examined the effects of non-myeloablative total body irradiation (TBI) in combination with immunosuppressive chemotherapy on immune homeostasis in rhesus macaques. Our results show that the administration of cyclosporin A or tacrolimus without radiotherapy did not result in lymphopenia. The addition of TBI to the regimen resulted in lymphopenia as well as alterations in the memory/naive ratio following reconstitution of lymphocyte populations. Dendritic cell (DC) numbers in whole blood were largely unaffected, while the monocyte population was altered by immunosuppressive treatment. Irradiation also resulted in increased levels of circulating cytokines and chemokines that correlated with T cell proliferative bursts and with the shift towards memory T cells. We also report that anti-thymocyte globulin (ATG) treatment and CD3 immunotoxin administration resulted in a selective and rapid depletion of naive CD4 and CD8 T cells and increased frequency of memory T cells. We also examined the impact of these treatments on reactivation of latent simian varicella virus (SVV) infection as a model of varicella zoster virus (VZV) infection of humans. None of the treatments resulted in overt SVV reactivation; however, select animals had transient increases in SVV-specific T cell responses following immunosuppression, suggestive of subclinical reactivation. Overall, we provide detailed observations into immune modulation by TBI and chemotherapeutic agents in rhesus macaques, an important research model of human disease. PMID:25902927

Melatonin-induced increase in sensitivity of human hepatocellular carcinoma cells to sorafenib is associated with reactive oxygen species production and mitophagy

PubMed Central

Prieto-Domínguez, Néstor; Ordóñez, Raquel; Fernández, Anna; Méndez-Blanco, Carolina; Baulies, Anna; Garcia-Ruiz, Carmen; Fernández-Checa, José C.; Mauriz, José L.; González-Gallego, Javier

2016-01-01

Effects of sorafenib in hepatocellular carcinoma (HCC) are frequently transient due to tumor-acquired resistance, a phenotype that could be targeted by other molecules to reduce this adaptive response. Because melatonin is known to exert antitumor effects in HCC cells, this study investigated whether and how melatonin reduces resistance to sorafenib. Susceptibility to sorafenib (10 nM to 50 μM) in the presence of melatonin (1 and 2 mM) was assessed in HCC cell lines HepG2, HuH7 and Hep3B. Cell viability was reduced by sorafenib from 1 μM in HepG2 or HuH7 cells, and 2.5 μM in Hep3B cells. Co-administration of melatonin and sorafenib exhibited a synergistic cytotoxic effect on HepG2 and HuH7 cells, while Hep3B cells displayed susceptibility to doses of sorafenib that had no effect when administrated alone. Co-administration of 2.5 μM sorafenib and 1 mM melatonin induced apoptosis in Hep3B cells, increasing PARP hydrolysis and BAX expression. We also observed an early colocalization of mitochondria with lysosomes, correlating with the expression of mitophagy markers PINK1 and Parkin and a reduction of mitofusin-2 and mtDNA compared with sorafenib administration alone. Moreover, increased reactive oxygen species production and mitochondrial membrane depolarization were elicited by drug combination, suggesting their contribution to mitophagy induction. Interestingly, Parkin silencing by siRNA to impair mitophagy significantly reduced cell killing, PARP cleavage and BAX expression. These results demonstrate that the pro-oxidant capacity of melatonin and its impact on mitochondria stability and turnover via mitophagy increase sensitivity to the cytotoxic effect of sorafenib. PMID:27484637

Trehalose Liposomes Suppress the Growth of Tumors on Human Lung Carcinoma-bearing Mice by Induction of Apoptosis In Vivo.

PubMed

Ichihara, Hideaki; Kuwabara, Keiji; Matsumoto, Yoko

2017-11-01

Previous evidence demonstrates that trehalose liposomes (DMTreC14) composed of L-α-dimyristoylphosphatidylcholine (DMPC) and α-D-glycopyranosyl-α-D-glucopyranoside monomyristate (TreC14) inhibit proliferation and invasion on lung carcinoma (A549 cells) in vitro. Here, we aimed to investigate suppressive effects of DMTreC14 on the growth of tumor on human lung carcinoma bearing mice. DMTreC14 composed of 30 mol% DMPC and 70 mol% TreC14 were prepared by the sonication method. Anti-tumor activities of DMTreC14 using the subcutaneous and orthotopic graft-bearing mice of A549 cells were investigated in vivo. The remarkable reduction of volume and weight in subcutaneous tumors on subcutaneous lung carcinoma-bearing mice topically administrated with DMTreC14 were obtained. Apoptotic-positive cells in the subcutaneous tumor slice of subcutaneous lung carcinoma-bearing mice topically administrated with DMTreC14 were observed using TUNEL staining. Lung weights on the orthotopic graft-bearing mice of lung carcinoma intravenously administrated with DMTreC14 were markedly decreased compared to those of the control group. Remarkable decrease in dimensions of tumor area of lung on the orthotopic graft-bearing mice of lung carcinoma intravenously administrated with DMTreC14 was obtained in histological analysis using the hematoxylin and eosin staining. Remarkably high anti-tumor activities of DMTreC14 for the subcutaneous and orthotopic graft-bearing mice of lung carcinoma accompanied with apoptosis were revealed for the first time in vivo. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

TCR-engineered, customized, antitumor T cells for cancer immunotherapy: advantages and limitations.

PubMed

Chhabra, Arvind

2011-01-05

The clinical outcome of the traditional adoptive cancer immunotherapy approaches involving the administration of donor-derived immune effectors, expanded ex vivo, has not met expectations. This could be attributed, in part, to the lack of sufficient high-avidity antitumor T-cell precursors in most cancer patients, poor immunogenicity of cancer cells, and the technological limitations to generate a sufficiently large number of tumor antigen-specific T cells. In addition, the host immune regulatory mechanisms and immune homeostasis mechanisms, such as activation-induced cell death (AICD), could further limit the clinical efficacy of the adoptively administered antitumor T cells. Since generation of a sufficiently large number of potent antitumor immune effectors for adoptive administration is critical for the clinical success of this approach, recent advances towards generating customized donor-specific antitumor-effector T cells by engrafting human peripheral blood-derived T cells with a tumor-associated antigen-specific transgenic T-cell receptor (TCR) are quite interesting. This manuscript provides a brief overview of the TCR engineering-based cancer immunotherapy approach, its advantages, and the current limitations.

77 FR 67379 - Draft Guidance for Industry and Food and Drug Administration Staff; Highly Multiplexed...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-11-09

... approaches. The document does not apply to devices that are intended to screen donors of blood and blood components, and donors of human cells, tissues, and cellular and tissue-based products for communicable...

Repeated Lentivirus-Mediated Granulocyte Colony-Stimulating Factor Administration to Treat Canine Cyclic Neutropenia

PubMed Central

Yanay, Ofer; Dale, David C.

2012-01-01

Abstract Cyclic neutropenia occurs in humans and gray collie dogs, is characterized by recurrent neutropenia, and is treated by repeated injections of recombinant granulocyte colony-stimulating factor (rG-CSF). As dose escalation of lentivirus may be clinically necessary, we monitored the outcome of four sequential intramuscular injections of G-CSF-lentivirus (3×107 IU/kg body weight) to a normal dog and a gray collie. In the normal dog absolute neutrophil counts were significantly increased after each dose of virus, with mean levels of 27.75±3.00, 31.50±1.40, 35.05±1.68, and 43.88±2.94×103 cells/μl, respectively (p<0.001), and elevated neutrophil counts of 31.18±7.81×103 cells/μl were maintained for more than 6 years with no adverse effects. A gray collie dog with a mean count of 1.94±1.48×103 cells/μl received G-CSF-lentivirus and we observed sustained elevations in neutrophil levels for more than 5 months with a mean of 26.00±11.00×103 cells/μl, significantly increased over the pretreatment level (p<0.001). After the second and third virus administrations mean neutrophil counts of 15.80±6.14 and 11.52±4.90×103 cells/μl were significantly reduced compared with cell counts after the first virus administration (p<0.001). However, after the fourth virus administration mean neutrophil counts of 15.21±4.50×103 cells/μl were significantly increased compared with the previous administration (p<0.05). Throughout the nearly 3 years of virus administrations the dog gained weight, was healthy, and showed neutrophil counts significantly higher than pretreatment levels (p<0.001). These studies suggest that patients with cyclic and other neutropenias may be treated with escalating doses of G-CSF-lentivirus to obtain a desired therapeutic neutrophil count. PMID:22845776

Mobilization of hematopoietic stem cells with highest self-renewal by G-CSF precedes clonogenic cell mobilization peak.

PubMed

Winkler, Ingrid G; Wiercinska, Eliza; Barbier, Valerie; Nowlan, Bianca; Bonig, Halvard; Levesque, Jean-Pierre

2016-04-01

Harvest of granulocyte colony-stimulating factor (G-CSF)-mobilized hematopoietic stem cells (HSCs) begins at day 5 of G-CSF administration, when most donors have achieved maximal mobilization. This is based on surrogate markers for HSC mobilization, such as CD34(+) cells and colony-forming activity in blood. However, CD34(+) cells or colony-forming units in culture (CFU-C) are heterogeneous cell populations with hugely divergent long-term repopulation potential on transplantation. HSC behavior is influenced by the vascular bed in the vicinity of which they reside. We hypothesized that G-CSF may mobilize sequentially cells proximal and more distal to bone marrow venous sinuses where HSCs enter the blood. We addressed this question with functional serial transplantation assays using blood and bone marrow after specific time points of G-CSF treatment in mice. We found that in mice, blood collected after only 48 hours of G-CSF administration was as enriched in serially reconstituting HSCs as blood collected at 5 days of G-CSF treatment. Similarly, mobilized Lin(-)CD34(+) cells were relatively enriched in more primitive Lin(-)CD34(+)CD38(-) cells at day 2 of G-CSF treatment compared with later points in half of human donors tested (n = 6). This suggests that in both humans and mice, hematopoietic progenitor and stem cells do not mobilize uniformly according to their maturation stage, with most potent HSCs mobilizing as early as day 2 of G-CSF. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 Attenuate Gardnerella vaginalis-Infected Bacterial Vaginosis in Mice.

PubMed

Jang, Se-Eun; Jeong, Jin-Ju; Choi, Su-Young; Kim, Hyunji; Han, Myung Joo; Kim, Dong-Hyun

2017-05-23

Oral administration of a probiotic mixture (PM; Respecta ® ) consisting of Lactobacillus rhamnosus HN001 (L1), Lactobacillus acidophilus La-14 (L2), and lactoferrin RCXTM results in colonization of these probiotics in the vagina of healthy women. Therefore, we examined whether vaginal colonization of the PM ingredients L1 and L2 could attenuate bacterial vaginosis (BV). BV was induced in mice via β-estradiol-3-benzoate-induced immunosuppression and intravaginal inoculation with Gardnerella vaginalis (GV). Inflammatory markers were analyzed using enzyme-linked immunosorbent assay, immunoblotting, quantitative polymerase chain reaction, and flow cytometry. Oral or intravaginal administration of PM resulted in colonization of L1 and L2 in the vagina. Oral or intravaginal administration of L1, L2, or PM significantly inhibited GV-induced epithelial cell disruption, myeloperoxidase activity, NF-κB activation, and IL-1β and TNF-α expression ( p < 0.05). Administration of these probiotics also inhibited IL-17 and RORγt expression but increased IL-10 and Foxp3 expression. Of these probiotics, L2 most effectively attenuated GV-induced BV, followed by L1 and PM. Oral administration was more effective against GV-induced BV than intravaginal administration. L1 and L2 also significantly inhibited the adherence of GV to HeLa cells (a human cervical cancer cell line) and GV growth in vitro. In addition, L1 and L2 inhibited lipopolysaccharide-induced NF-κB activation in macrophages and the differentiation of splenocytes into Th17 cells in vitro, but increased their differentiation into Treg cells. Our study suggests that L1, L2, and PM attenuated GV-induced vaginosis by regulating both vaginal and systemic innate and adaptive immune responses rather than direct competition or killing of GV in the vagina.

Possibilities of vaccine manufacture in human diploid cell strains with a serum replacement factor.

PubMed

Candal, F J; George, V G; Ades, E W

1991-07-01

Cell lines MDCK (canine kidney), BGM (Buffalo green monkey kidney) and human embryonic lung fibroblast will support viral growth efficiently in medium without serum. Both MRC-5 and WI-38 cell strains have been approved by the Food and Drug Administration for manufacturing viral vaccines against cytomegalovirus and varicella-zoster virus. In this study we examine these two cell lines and viruses for their ability to grow in medium containing a serum replacement. The serum substitute used is LPSR-1 (low protein serum replacement). Using LPSR-1 in defined medium, we demonstrate multipassage cell growth and viral cultivation and replication equivalent to those obtained in medium containing fetal bovine serum (FBS). Viral growth after complete elimination of FBS varies and depends on cell line and virus. Serum substitutes can eliminate FBS in the viral growth phase of vaccine production and reduce costs.

Administration of interleukin-12 enhances the therapeutic efficacy of dendritic cell-based tumor vaccines in mouse hepatocellular carcinoma.

PubMed

Tatsumi, T; Takehara, T; Kanto, T; Miyagi, T; Kuzushita, N; Sugimoto, Y; Jinushi, M; Kasahara, A; Sasaki, Y; Hori, M; Hayashi, N

2001-10-15

Dendritic cells (DCs) are potent antigen-presenting cells that are capable of priming systemic antitumor immune response. Here, we evaluated the combined effectiveness of tumor lysate-pulsed DC immunization and interleukin (IL)-12 administration on the induction of antitumor immunity in a mouse hepatocellular carcinoma (HCC) model. Mouse DCs were pulsed with lysate of BNL 1ME A.7R.1 (BNL), a BALB/c-derived HCC cell line, and then injected into syngeneic mice in combination with systemic administration of IL-12. Lymphocytes from mice treated with BNL lysate-pulsed DCs and IL-12 showed stronger cytolytic activity and produced higher amounts of IFN-gamma than those from mice treated with BNL lysate-pulsed DCs alone. Although immunization with BNL lysate-pulsed DCs alone did not lead to complete regression of established tumors, it significantly inhibited tumor growth compared with vehicle injection. Importantly, the combined therapy of BNL lysate-pulsed DCs and IL-12 resulted in tumor rejection or significant inhibition of tumor growth compared with mice treated with BNL lysate-pulsed DCs alone. In vivo lymphocyte depletion experiments demonstrated that this combination was dependent on both CD8+ and CD4+ T cells, but not natural killer cells. These results demonstrated that IL-12 administration enhanced the therapeutic effect of immunization of tumor lysate-pulsed DCs against HCC in mice. This combination of IL-12 and DCs may be useful for suppressing the growth of residual tumor after primary therapy of human HCC.

Sequentially administrated of pemetrexed with icotinib/erlotinib in lung adenocarcinoma cell lines in vitro

PubMed Central

Feng, Xiuli; Zhang, Yan; Li, Tao; Li, Yu

2017-01-01

Combination of chemotherapy and epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) had been proved to be a potent anti-drug for the treatment of tumors. However, survival time was not extended for the patients with lung adenocarcinoma (AdC) compared with first-line chemotherapy. In the present study, we attempt to assess the optimal schedule of the combined administration of pemetrexed and icotinib/erlotinib in AdC cell lines. Human lung AdC cell lines with wild-type (A549), EGFR T790M (H1975) and activating EGFR mutation (HCC827) were applied in vitro to assess the differential efficacy of various sequential regimens on cell viability, cell apoptosis and cell cycle distribution. The results suggested that the antiproliferative effect of the sequence of pemetrexed followed by icotinib/erlotinib was more effective than that of icotinib/erlotinib followed by pemetrexed. Additionally, a reduction of G1 phase and increased S phase in sequence of pemetrexed followed by icotinib/erlotinib was also observed, promoting cell apoptosis. Thus, the sequential administration of pemetrexed followed by icotinib/erlotinib exerted a synergistic effect on HCC827 and H1975 cell lines compared with the reverse sequence. The sequential treatment of pemetrexed followed by icotinib/erlotinib has been demonstrated promising results. This treatment strategy warrants further confirmation in patients with advanced lung AdC. PMID:29371987

Sequentially administrated of pemetrexed with icotinib/erlotinib in lung adenocarcinoma cell lines in vitro.

PubMed

Feng, Xiuli; Zhang, Yan; Li, Tao; Li, Yu

2017-12-26

Combination of chemotherapy and epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) had been proved to be a potent anti-drug for the treatment of tumors. However, survival time was not extended for the patients with lung adenocarcinoma (AdC) compared with first-line chemotherapy. In the present study, we attempt to assess the optimal schedule of the combined administration of pemetrexed and icotinib/erlotinib in AdC cell lines. Human lung AdC cell lines with wild-type (A549), EGFR T790M (H1975) and activating EGFR mutation (HCC827) were applied in vitro to assess the differential efficacy of various sequential regimens on cell viability, cell apoptosis and cell cycle distribution. The results suggested that the antiproliferative effect of the sequence of pemetrexed followed by icotinib/erlotinib was more effective than that of icotinib/erlotinib followed by pemetrexed. Additionally, a reduction of G1 phase and increased S phase in sequence of pemetrexed followed by icotinib/erlotinib was also observed, promoting cell apoptosis. Thus, the sequential administration of pemetrexed followed by icotinib/erlotinib exerted a synergistic effect on HCC827 and H1975 cell lines compared with the reverse sequence. The sequential treatment of pemetrexed followed by icotinib/erlotinib has been demonstrated promising results. This treatment strategy warrants further confirmation in patients with advanced lung AdC.

Bioactivity and Safety of IL13Rα2-Redirected Chimeric Antigen Receptor CD8+ T Cells in Patients with Recurrent Glioblastoma.

PubMed

Brown, Christine E; Badie, Behnam; Barish, Michael E; Weng, Lihong; Ostberg, Julie R; Chang, Wen-Chung; Naranjo, Araceli; Starr, Renate; Wagner, Jamie; Wright, Christine; Zhai, Yubo; Bading, James R; Ressler, Julie A; Portnow, Jana; D'Apuzzo, Massimo; Forman, Stephen J; Jensen, Michael C

2015-09-15

A first-in-human pilot safety and feasibility trial evaluating chimeric antigen receptor (CAR)-engineered, autologous primary human CD8(+) cytotoxic T lymphocytes (CTL) targeting IL13Rα2 for the treatment of recurrent glioblastoma (GBM). Three patients with recurrent GBM were treated with IL13(E13Y)-zetakine CD8(+) CTL targeting IL13Rα2. Patients received up to 12 local infusions at a maximum dose of 10(8) CAR-engineered T cells via a catheter/reservoir system. We demonstrate the feasibility of manufacturing sufficient numbers of autologous CTL clones expressing an IL13(E13Y)-zetakine CAR for redirected HLA-independent IL13Rα2-specific effector function for a cohort of patients diagnosed with GBM. Intracranial delivery of the IL13-zetakine(+) CTL clones into the resection cavity of 3 patients with recurrent disease was well-tolerated, with manageable temporary brain inflammation. Following infusion of IL13-zetakine(+) CTLs, evidence for transient anti-glioma responses was observed in 2 of the patients. Analysis of tumor tissue from 1 patient before and after T-cell therapy suggested reduced overall IL13Rα2 expression within the tumor following treatment. MRI analysis of another patient indicated an increase in tumor necrotic volume at the site of IL13-zetakine(+) T-cell administration. These findings provide promising first-in-human clinical experience for intracranial administration of IL13Rα2-specific CAR T cells for the treatment of GBM, establishing a foundation on which future refinements of adoptive CAR T-cell therapies can be applied. ©2015 American Association for Cancer Research.

MSCs ameliorates DPN induced cellular pathology via [Ca2+ ]i homeostasis and scavenging the pro-inflammatory cytokines.

PubMed

Chandramoorthy, Harish C; Bin-Jaliah, Ismaeel; Karari, Hussian; Rajagopalan, Prasanna; Ahmed Shariff, Mohammed Eajaz; Al-Hakami, Ahmed; Al-Humayad, Suliman M; Baptain, Fawzi A; Ahmed, Humeda Suekit; Yassin, Hanaa Z; Haidara, Mohamed A

2018-02-01

The MSCs of various origins are known to ameliorate or modulate cell survival strategies. We investigated, whether UCB MSCs could improve the survival of the human neuronal cells and/or fibroblast assaulted with DPN sera. The results showed, the co-culture of UCB MSCs with human neuronal cells and/or fibroblasts could effectively scavenge the pro-inflammatory cytokines TNF-α, IL-1β, IFN-ɤ and IL - 12 and control the pro-apoptotic expression of p53/Bax. Further co-culture of UCB MSCs have shown to induce anti-inflammatory cytokines like IL-4, IL-10 and TGF-β and anti-apoptotic Bclxl/Bcl2 expression in the DPN sera stressed cells. Amelioration of elevated [Ca 2+ ] i and cROS, the portent behind the NFκB/Caspase-3 mediated inflammation in DPN rescued the cells from apoptosis. The results of systemic administration of BM MSCs improved DPN pathology in rat as extrapolated from human cell model. The BM MSCs ameliorated prolonged distal motor latency (control: 0.70 ± 0.06, DPN: 1.29 ± 0.13 m/s DPN + BM MSCs: 0.89 ± 0.02 m/s, p < 0.05) and lowered high amplitude of compound muscle action potentials (CMAPs) (control: 12.36 ± 0.41, DPN: 7.52 ± 0.61 mV, DPN + MSCs: 8.79 ± 0.53 mV, p < 0.05), while slowly restoring the plasma glucose levels. Together, all these results showed that administration of BM or UCB MSCs improved the DPN via ameliorating pro-inflammatory cytokine signaling and [Ca 2+ ] i homeostasis. © 2017 Wiley Periodicals, Inc.

Administration of CD34+ cells after stroke enhances neurogenesis via angiogenesisin a mouse model

PubMed Central

Taguchi, Akihiko; Soma, Toshihiro; Tanaka, Hidekazu; Kanda, Takayoshi; Nishimura, Hiroyuki; Yoshikawa, Hiroo; Tsukamoto, Yoshitane; Iso, Hiroyuki; Fujimori, Yoshihiro; Stern, David M.; Naritomi, Hiroaki; Matsuyama, Tomohiro

2004-01-01

Thrombo-occlusive cerebrovascular disease resulting in stroke and permanent neuronal loss is an important cause of morbidity and mortality. Because of the unique properties of cerebral vasculature and the limited reparative capability of neuronal tissue, it has been difficult to devise effective neuroprotective therapies in cerebral ischemia. Our results demonstrate that systemic administration of human cord blood–derived CD34+ cells to immunocompromised mice subjected to stroke 48 hours earlier induces neovascularization in the ischemic zone and provides a favorable environment for neuronal regeneration. Endogenous neurogenesis, suppressed by an antiangiogenic agent, is accelerated as a result of enhanced migration of neuronal progenitor cells to the damaged area, followed by their maturation and functional recovery. Our data suggest an essential role for CD34+ cells in promoting directly or indirectly an environment conducive to neovascularization of ischemic brain so that neuronal regeneration can proceed. PMID:15286799

The role of the obestatin/GPR39 system in human gastric adenocarcinomas.

PubMed

Alén, Begoña O; Leal-López, Saúl; Alén, María Otero; Viaño, Patricia; García-Castro, Victoria; Mosteiro, Carlos S; Beiras, Andrés; Casanueva, Felipe F; Gallego, Rosalía; García-Caballero, Tomás; Camiña, Jesús P; Pazos, Yolanda

2016-02-02

Obestatin, a 23-amino acid peptide encoded by the ghrelin gene, and the GPR39 receptor were reported to be involved in the control of mitogenesis of gastric cancer cell lines; however, the relationship between the obestatin/GPR39 system and gastric cancer progression remains unknown. In the present study, we determined the expression levels of the obestatin/GPR39 system in human gastric adenocarcinomas and explored their potential functional roles. Twenty-eight patients with gastric adenocarcinomas were retrospectively studied, and clinical data were obtained. The role of obestatin/GPR39 in gastric cancer progression was studied in vitro using the human gastric adenocarcinoma AGS cell line. Obestatin exogenous administration in these GPR39-bearing cells deregulated the expression of several hallmarks of the epithelial-mesenchymal transition (EMT) and angiogenesis. Moreover, obestatin signaling promoted phenotypic changes via GPR39, increasingly impacting on the cell morphology, proliferation, migration and invasion of these cells. In healthy human stomachs, obestatin expression was observed in the neuroendocrine cells and GPR39 expression was localized mainly in the chief cells of the oxyntic glands. In human gastric adenocarcinomas, no obestatin expression was found; however, an aberrant pattern of GPR39 expression was discovered, correlating to the dedifferentiation of the tumor. Altogether, our data strongly suggest the involvement of the obestatin/GPR39 system in the pathogenesis and/or clinical outcome of human gastric adenocarcinomas and highlight the potential usefulness of GPR39 as a prognostic marker in gastric cancer.

The role of the obestatin/GPR39 system in human gastric adenocarcinomas

PubMed Central

Alén, Begoña O.; Leal-López, Saúl; Alén, María Otero; Viaño, Patricia; García-Castro, Victoria; Mosteiro, Carlos S.; Beiras, Andrés; Casanueva, Felipe F.; Gallego, Rosalía; García-Caballero, Tomás; Camiña, Jesús P.; Pazos, Yolanda

2016-01-01

Obestatin, a 23-amino acid peptide encoded by the ghrelin gene, and the GPR39 receptor were reported to be involved in the control of mitogenesis of gastric cancer cell lines; however, the relationship between the obestatin/GPR39 system and gastric cancer progression remains unknown. In the present study, we determined the expression levels of the obestatin/GPR39 system in human gastric adenocarcinomas and explored their potential functional roles. Twenty-eight patients with gastric adenocarcinomas were retrospectively studied, and clinical data were obtained. The role of obestatin/GPR39 in gastric cancer progression was studied in vitro using the human gastric adenocarcinoma AGS cell line. Obestatin exogenous administration in these GPR39-bearing cells deregulated the expression of several hallmarks of the epithelial-mesenchymal transition (EMT) and angiogenesis. Moreover, obestatin signaling promoted phenotypic changes via GPR39, increasingly impacting on the cell morphology, proliferation, migration and invasion of these cells. In healthy human stomachs, obestatin expression was observed in the neuroendocrine cells and GPR39 expression was localized mainly in the chief cells of the oxyntic glands. In human gastric adenocarcinomas, no obestatin expression was found; however, an aberrant pattern of GPR39 expression was discovered, correlating to the dedifferentiation of the tumor. Altogether, our data strongly suggest the involvement of the obestatin/GPR39 system in the pathogenesis and/or clinical outcome of human gastric adenocarcinomas and highlight the potential usefulness of GPR39 as a prognostic marker in gastric cancer. PMID:26716511

Calcium alginate gels as stem cell matrix-making paracrine stem cell activity available for enhanced healing after surgery.

PubMed

Schmitt, Andreas; Rödel, Philipp; Anamur, Cihad; Seeliger, Claudine; Imhoff, Andreas B; Herbst, Elmar; Vogt, Stephan; van Griensven, Martijn; Winter, Gerhard; Engert, Julia

2015-01-01

Regeneration after surgery can be improved by the administration of anabolic growth factors. However, to locally maintain these factors at the site of regeneration is problematic. The aim of this study was to develop a matrix system containing human mesenchymal stem cells (MSCs) which can be applied to the surgical site and allows the secretion of endogenous healing factors from the cells. Calcium alginate gels were prepared by a combination of internal and external gelation. The gelling behaviour, mechanical stability, surface adhesive properties and injectability of the gels were investigated. The permeability of the gels for growth factors was analysed using bovine serum albumin and lysozyme as model proteins. Human MSCs were isolated, cultivated and seeded into the alginate gels. Cell viability was determined by AlamarBlue assay and fluorescence microscopy. The release of human VEGF and bFGF from the cells was determined using an enzyme-linked immunoassay. Gels with sufficient mechanical properties were prepared which remained injectable through a syringe and solidified in a sufficient time frame after application. Surface adhesion was improved by the addition of polyethylene glycol 300,000 and hyaluronic acid. Humans MSCs remained viable for the duration of 6 weeks within the gels. Human VEGF and bFGF was found in quantifiable concentrations in cell culture supernatants of gels loaded with MSCs and incubated for a period of 6 weeks. This work shows that calcium alginate gels can function as immobilization matrices for human MSCs.

Engineering human T cells for resistance to methotrexate and mycophenolate mofetil as an in vivo cell selection strategy.

PubMed

Jonnalagadda, Mahesh; Brown, Christine E; Chang, Wen-Chung; Ostberg, Julie R; Forman, Stephen J; Jensen, Michael C

2013-01-01

Gene transfer and drug selection systems that enforce ongoing transgene expression in vitro and in vivo which are compatible with human pharmaceutical drugs are currently underdeveloped. Here, we report on the utility of incorporating human enzyme muteins that confer resistance to the lymphotoxic/immunosuppressive drugs methotrexate (MTX) and mycophenolate mofetil (MMF) in a multicistronic lentiviral vector for in vivo T lymphocyte selection. We found that co-expression of human dihydrofolate reductase (DHFR(FS); L22F, F31S) and inosine monophosphate dehydrogenase II (IMPDH2(IY); T333I, S351Y) conferred T cell resistance to the cytocidal and anti-proliferative effects of these drugs at concentrations that can be achieved clinically (up to 0.1 µM MTX and 1.0 µM MPA). Furthermore, using a immunodeficient mouse model that supports the engraftment of central memory derived human T cells, in vivo selection studies demonstrate that huEGFRt(+)DHFR(FS+)IMPDH2(IY+) T cells could be enriched following adoptive transfer either by systemic administration of MTX alone (4.4 -fold), MMF alone (2.9-fold), or combined MTX and MMF (4.9-fold). These findings demonstrate the utility of both DHFR(FS)/MTX and IMPDH2(IY)/MMF for in vivo selection of lentivirally transduced human T cells. Vectors incorporating these muteins in combination with other therapeutic transgenes may facilitate the selective engraftment of therapeutically active cells in recipients.

Administration of herpes simplex-thymidine kinase-expressing donor T cells with a T-cell-depleted allogeneic marrow graft.

PubMed

Tiberghien, P; Ferrand, C; Lioure, B; Milpied, N; Angonin, R; Deconinck, E; Certoux, J M; Robinet, E; Saas, P; Petracca, B; Juttner, C; Reynolds, C W; Longo, D L; Hervé, P; Cahn, J Y

2001-01-01

Administration of donor T cells expressing the herpes simplex-thymidine kinase (HS-tk) with a hematopoietic stem cell (HSC) transplantation could allow, if graft-versus-host disease (GVHD) was to occur, a selective in vivo depletion of these T cells by the use of ganciclovir (GCV). The study evaluates the feasibility of such an approach. Escalating numbers of donor HS-tk-expressing CD3(+) gene-modified cells (GMCs) are infused with a T-cell-depleted bone marrow transplantation (BMT). Twelve patients with hematological malignancies received 2 x 10(5) (n = 5), 6 x 10(5) (n = 5), or 20 x 10(5) (n = 2) donor CD3(+) GMCs/kg with a BMT from a human leukocyte antigen (HLA)-identical sibling. No acute toxicity was associated with GMC administration. An early increase of circulating GMCs followed by a progressive decrease and long-lasting circulation of GMCs was documented. GCV treatment resulted in significant rapid decrease in circulating GMCs. Three patients developed acute GVHD, with a grade of at least II, while one patient developed chronic GVHD. Treatment with GCV alone was associated with a complete remission (CR) in 2 patients with acute GVHD, while the addition of glucocorticoids was necessary to achieve a CR in the last case. Long-lasting CR occurred with GCV treatment in the patient with chronic GVHD. Unfortunately, Epstein-Barr virus-lymphoproliferative disease occurred in 3 patients. Overall, the administration of low numbers of HS-tk-expressing T cells early following an HLA-identical BMT is associated with no acute toxicity, persistent circulation of the GMCs, and GCV-sensitive GVHD. Such findings open the way to the infusion of higher numbers of gene-modified donor T cells to enhance post-BMT immune competence while preserving GCV-sensitive alloreactivity.

Hsp25 and Hsp70 in rodent tumors treated with doxorubicin and lovastatin

PubMed Central

Ciocca, Daniel R.; Rozados, Viviana R.; Carrión, F. Darío Cuello; Gervasoni, Silvia I.; Matar, Pablo; Scharovsky, O. Graciela

2003-01-01

Heat shock protein 27 (Hsp27) and Hsp70 have been involved in resistance to anticancer drugs in human breast cancer cells growing in vitro and in vivo. In this study, we examined the expression of Hsp25 (the rodent homologue to human Hsp27) and Hsp70 in 3 different rodent tumors (a mouse breast carcinoma, a rat sarcoma, and a rat lymphoma maintained by subcutaneous passages) treated in vivo with doxorubicin (DOX) and lovastatin (LOV). All tumors showed massive cell death under control untreated conditions, and this massive death increased after cytotoxic drug administration. In this study, we show that this death was due to classic apoptosis. The tumors also showed isolated apoptotic cells between viable tumor cells, and this occurred more significantly in the lymphoma. The tumor type that was more resistant to cell death was the sarcoma, and this was found in sarcomas growing both under control conditions and after cytotoxic drug administration. Moreover, sarcomas showed the highest expression levels of Hsp25 in the viable tumor cells growing under untreated conditions, and these levels increased after DOX and LOV administration. After drug treatment, only sarcoma tumor cells showed a significant increase in Hsp70. In other words, sarcomas were the tumors with lower cell death, displayed a competent Hsp70 and Hsp25 response with nuclear translocation, and had the highest levels of Hsp25. In sarcomas, Hsp25 and Hsp70 were found in viable tumor cells located around the blood vessels, and these areas showed the most resistant tumor cell phenotype after chemotherapy. In addition, Hsp25 expression was found in endothelial cells as unique feature revealed only in lymphomas. In conclusion, our study shows that each tumor type has unique features regarding the expression of Hsp25 and Hsp70 and that these proteins seem to be implicated in drug resistance mainly in sarcomas, making these model systems important to perform more mechanistic studies on the role of Hsps in resistance to certain cytotoxic drugs. PMID:12820652

Long-term administration of inulin-type fructans has no significant lipid-lowering effect in normolipidemic humans.

PubMed

Forcheron, Fabien; Beylot, Michel

2007-08-01

Short-term studies have shown that the addition to diet of inulin-type fructans, a nondigestible carbohydrate, may have a plasma lipid-lowering effect in humans. Whether this beneficial effect persists during long-term administration has not been determined. The study was aimed at determining whether a prolonged (6 months) administration of inulin-type fructans to healthy subjects has a lipid-lowering action. In a double-blind, randomized, placebo-controlled study, 17 healthy subjects were studied before and after 6 months of daily administration of placebo (8 subjects) or 10 g of a mix of inulin and oligofructose (9 subjects). During this 6-month period, they consumed their usual diet and did not modify their everyday way of life. We measured plasma lipid concentrations; cholesterol synthesis and hepatic lipogenesis; and adipose tissue and circulating mononuclear cell messenger RNA concentrations of key regulatory genes of cholesterol metabolism. Compared with the administration of placebo, the administration of inulin-type fructans had no effect on plasma triacylglycerol concentrations and hepatic lipogenesis and induced only a nonsignificant trend for decreased plasma total and low-density lipoprotein cholesterol levels and increased high-density lipoprotein cholesterol concentration. Cholesterol synthesis was not significantly modified. Of all the messenger RNA concentrations measured, none was significantly modified by the administration of inulin-type fructans. In conclusion, contrary to what was observed in short-term studies, we observed no significant beneficial effect of a long-term (6-month) administration of inulin-type fructans on plasma lipids in healthy human subjects.

21 CFR 610.11 - General safety.

Code of Federal Regulations, 2012 CFR

2012-04-01

... Whole Blood, Red Blood Cells, Cryoprecipitated AHF, Platelets, Plasma, or Cellular Therapy Products. (2... for administration to humans. The general safety test is required in addition to other specific tests... shall be performed as specified in this section, unless: Modification is prescribed in the additional...

21 CFR 610.11 - General safety.

Code of Federal Regulations, 2013 CFR

2013-04-01

... Whole Blood, Red Blood Cells, Cryoprecipitated AHF, Platelets, Plasma, or Cellular Therapy Products. (2... for administration to humans. The general safety test is required in addition to other specific tests... shall be performed as specified in this section, unless: Modification is prescribed in the additional...

21 CFR 610.11 - General safety.

Code of Federal Regulations, 2014 CFR

2014-04-01

... Whole Blood, Red Blood Cells, Cryoprecipitated AHF, Platelets, Plasma, or Cellular Therapy Products. (2... for administration to humans. The general safety test is required in addition to other specific tests... shall be performed as specified in this section, unless: Modification is prescribed in the additional...

21 CFR 610.11 - General safety.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Whole Blood, Red Blood Cells, Cryoprecipitated AHF, Platelets, Plasma, or Cellular Therapy Products. (2... for administration to humans. The general safety test is required in addition to other specific tests... shall be performed as specified in this section, unless: Modification is prescribed in the additional...

Potent In Vivo NK Cell-Mediated Elimination of HIV-1-Infected Cells Mobilized by a gp120-Bispecific and Hexavalent Broadly Neutralizing Fusion Protein

PubMed Central

Bardhi, Ariola; Wu, Yanling; Chen, Weizao; Li, Wei; Zhu, Zhongyu; Zheng, Jian Hua; Wong, Hing; Jeng, Emily; Jones, Jennifer; Ochsenbauer, Christina; Kappes, John C.; Dimitrov, Dimiter S.; Ying, Tianlei

2017-01-01

ABSTRACT Antibodies bound to human immunodeficiency virus type 1 (HIV-1) envelope protein expressed by infected cells mobilize antibody-dependent cellular cytotoxicity (ADCC) to eliminate the HIV-1-infected cells and thereby suppress HIV-1 infection and delay disease progression. Studies treating HIV-1-infected individuals with latency reactivation agents to reduce their latent HIV-1 reservoirs indicated that their HIV-1-specific immune responses were insufficient to effectively eliminate the reactivated latent HIV-1-infected T cells. Mobilization of ADCC may facilitate elimination of reactivated latent HIV-1-infected cells to deplete the HIV-1 reservoir and contribute to a functional HIV-1 cure. The most effective antibodies for controlling and eradicating HIV-1 infection would likely have the dual capacities of potently neutralizing a broad range of HIV-1 isolates and effectively mobilizing HIV-1-specific ADCC to eliminate HIV-1-infected cells. For this purpose, we constructed LSEVh-LS-F, a broadly neutralizing, defucosylated hexavalent fusion protein specific for both the CD4 and coreceptor gp120-binding sites. LSEVh-LS-F potently inhibited in vivo HIV-1 and simian-human immunodeficiency virus (SHIV) infection in humanized mouse and macaque models, respectively, including in vivo neutralization of HIV-1 strains resistant to the broadly neutralizing antibodies VRC01 and 3BNC117. We developed a novel humanized mouse model to evaluate in vivo human NK cell-mediated elimination of HIV-1-infected cells by ADCC and utilized it to demonstrate that LSEVh-LS-F rapidly mobilized NK cells to eliminate >80% of HIV-1-infected cells in vivo 1 day after its administration. The capacity of LSEVh-LS-F to eliminate HIV-1-infected cells via ADCC combined with its broad neutralization activity supports its potential use as an immunotherapeutic agent to eliminate reactivated latent cells and deplete the HIV-1 reservoir. IMPORTANCE Mobilization of antibody-dependent cellular cytotoxicity (ADCC) to eliminate reactivated latent HIV-1-infected cells is a strategy which may contribute to depleting the HIV-1 reservoir and achieving a functional HIV-1 cure. To more effectively mobilize ADCC, we designed and constructed LSEVh-LS-F, a broadly neutralizing, defucosylated hexavalent fusion protein specific for both the CD4 and coreceptor gp120-binding sites. LSEVh-LS-F potently inhibited in vivo HIV-1 and SHIV infection in humanized mouse and macaque models, respectively, including in vivo neutralization of an HIV-1 strain resistant to the broadly neutralizing antibodies VRC01 and 3BNC117. Using a novel humanized mouse model, we demonstrated that LSEVh-LS-F rapidly mobilized NK cells to eliminate >80% of HIV-1-infected cells in vivo 1 day after its administration. The capacity of LSEVh-LS-F to eliminate HIV-1-infected cells via ADCC combined with its broad neutralization activity supports its potential use as an immunotherapeutic agent to eliminate reactivated latent cells and deplete the HIV-1 reservoir. PMID:28794022

Potent In Vivo NK Cell-Mediated Elimination of HIV-1-Infected Cells Mobilized by a gp120-Bispecific and Hexavalent Broadly Neutralizing Fusion Protein.

PubMed

Bardhi, Ariola; Wu, Yanling; Chen, Weizao; Li, Wei; Zhu, Zhongyu; Zheng, Jian Hua; Wong, Hing; Jeng, Emily; Jones, Jennifer; Ochsenbauer, Christina; Kappes, John C; Dimitrov, Dimiter S; Ying, Tianlei; Goldstein, Harris

2017-10-15

Antibodies bound to human immunodeficiency virus type 1 (HIV-1) envelope protein expressed by infected cells mobilize antibody-dependent cellular cytotoxicity (ADCC) to eliminate the HIV-1-infected cells and thereby suppress HIV-1 infection and delay disease progression. Studies treating HIV-1-infected individuals with latency reactivation agents to reduce their latent HIV-1 reservoirs indicated that their HIV-1-specific immune responses were insufficient to effectively eliminate the reactivated latent HIV-1-infected T cells. Mobilization of ADCC may facilitate elimination of reactivated latent HIV-1-infected cells to deplete the HIV-1 reservoir and contribute to a functional HIV-1 cure. The most effective antibodies for controlling and eradicating HIV-1 infection would likely have the dual capacities of potently neutralizing a broad range of HIV-1 isolates and effectively mobilizing HIV-1-specific ADCC to eliminate HIV-1-infected cells. For this purpose, we constructed LSEVh-LS-F, a broadly neutralizing, defucosylated hexavalent fusion protein specific for both the CD4 and coreceptor gp120-binding sites. LSEVh-LS-F potently inhibited in vivo HIV-1 and simian-human immunodeficiency virus (SHIV) infection in humanized mouse and macaque models, respectively, including in vivo neutralization of HIV-1 strains resistant to the broadly neutralizing antibodies VRC01 and 3BNC117. We developed a novel humanized mouse model to evaluate in vivo human NK cell-mediated elimination of HIV-1-infected cells by ADCC and utilized it to demonstrate that LSEVh-LS-F rapidly mobilized NK cells to eliminate >80% of HIV-1-infected cells in vivo 1 day after its administration. The capacity of LSEVh-LS-F to eliminate HIV-1-infected cells via ADCC combined with its broad neutralization activity supports its potential use as an immunotherapeutic agent to eliminate reactivated latent cells and deplete the HIV-1 reservoir. IMPORTANCE Mobilization of antibody-dependent cellular cytotoxicity (ADCC) to eliminate reactivated latent HIV-1-infected cells is a strategy which may contribute to depleting the HIV-1 reservoir and achieving a functional HIV-1 cure. To more effectively mobilize ADCC, we designed and constructed LSEVh-LS-F, a broadly neutralizing, defucosylated hexavalent fusion protein specific for both the CD4 and coreceptor gp120-binding sites. LSEVh-LS-F potently inhibited in vivo HIV-1 and SHIV infection in humanized mouse and macaque models, respectively, including in vivo neutralization of an HIV-1 strain resistant to the broadly neutralizing antibodies VRC01 and 3BNC117. Using a novel humanized mouse model, we demonstrated that LSEVh-LS-F rapidly mobilized NK cells to eliminate >80% of HIV-1-infected cells in vivo 1 day after its administration. The capacity of LSEVh-LS-F to eliminate HIV-1-infected cells via ADCC combined with its broad neutralization activity supports its potential use as an immunotherapeutic agent to eliminate reactivated latent cells and deplete the HIV-1 reservoir. Copyright © 2017 American Society for Microbiology.

Metabolomic Analyses of Blood Plasma after Oral Administration of D-Glucosamine Hydrochloride to Dogs

PubMed Central

Osaki, Tomohiro; Azuma, Kazuo; Kurozumi, Seiji; Takamori, Yoshimori; Tsuka, Takeshi; Imagawa, Tomohiro; Okamoto, Yoshiharu; Minami, Saburo

2012-01-01

D-Glucosamine hydrochloride (GlcN∙HCl) is an endogenous amino monosaccharide synthesized from glucose that is useful in the treatment of joint diseases in both humans and animals. The aim of this study was to examine amino acid metabolism in dogs after oral administration of GlcN∙HCl. Accelerated fumarate respiration and elevated plasma levels of lactic acid and alanine were observed after administration. These results suggest that oral administration of GlcN∙HCl induces anaerobic respiration and starvation in cells, and we hypothesize that these conditions promote cartilage regeneration. Further studies are required to evaluate the expression of transforming growth factor-beta (TGF-β). PMID:23015778

Dose-Dependent Effect of Intravenous Administration of Human Umbilical Cord-Derived Mesenchymal Stem Cells in Neonatal Stroke Mice

PubMed Central

Tanaka, Emi; Ogawa, Yuko; Mukai, Takeo; Sato, Yoshiaki; Hamazaki, Takashi; Nagamura-Inoue, Tokiko; Harada-Shiba, Mariko; Shintaku, Haruo; Tsuji, Masahiro

2018-01-01

Neonatal brain injury induced by stroke causes significant disability, including cerebral palsy, and there is no effective therapy for stroke. Recently, mesenchymal stem cells (MSCs) have emerged as a promising tool for stem cell-based therapies. In this study, we examined the safety and efficacy of intravenously administered human umbilical cord-derived MSCs (UC-MSCs) in neonatal stroke mice. Pups underwent permanent middle cerebral artery occlusion at postnatal day 12 (P12), and low-dose (1 × 104) or high-dose (1 × 105) UC-MSCs were administered intravenously 48 h after the insult (P14). To evaluate the effect of the UC-MSC treatment, neurological behavior and cerebral blood flow were measured, and neuroanatomical analysis was performed at P28. To investigate the mechanisms of intravenously injected UC-MSCs, systemic blood flowmetry, in vivo imaging and human brain-derived neurotrophic factor (BDNF) measurements were performed. Functional disability was significantly improved in the high-dose UC-MSC group when compared with the vehicle group, but cerebral blood flow and cerebral hemispheric volume were not restored by UC-MSC therapy. The level of exogenous human BDNF was elevated only in the cerebrospinal fluid of one pup 24 h after UC-MSC injection, and in vivo imaging revealed that most UC-MSCs were trapped in the lungs and disappeared in a week without migration toward the brain or other organs. We found that systemic blood flow was stable over the 10 min after cell administration and that there were no differences in mortality among the groups. Immunohistopathological assessment showed that the percent area of Iba1-positive staining in the peri-infarct cortex was significantly reduced with the high-dose UC-MSC treatment compared with the vehicle treatment. These results suggest that intravenous administration of UC-MSCs is safe for a mouse model of neonatal stroke and improves dysfunction after middle cerebral artery occlusion by modulating the microglial reaction in the peri-infarct cortex. PMID:29568282

Homing of human B cells to lymphoid organs and B-cell lymphoma engraftment are controlled by cell adhesion molecule JAM-C.

PubMed

Doñate, Carmen; Ody, Christiane; McKee, Thomas; Ruault-Jungblut, Sylvie; Fischer, Nicolas; Ropraz, Patricia; Imhof, Beat A; Matthes, Thomas

2013-01-15

Junctional adhesion molecule C (JAM-C) is expressed by vascular endothelium and human but not mouse B lymphocytes. The level of JAM-C expression defines B-cell differentiation stages and allows the classification of marginal zone-derived (JAM-C-positive) and germinal center-derived (JAM-C-negative) B-cell lymphomas. In the present study, we investigated the role of JAM-C in homing of human B cells, using a xenogeneic nonobese diabetic/severe combined immunodeficient mouse model. Treatment with anti-JAM-C antibodies in short-term experiments reduced migration of normal and malignant JAM-C-expressing B cells to bone marrow, lymph nodes, and spleen. Blocking homing to the spleen is remarkable, as most other antiadhesion antibodies reduce homing of B cells only to bone marrow and lymph nodes. Long-term administration of anti-JAM-C antibodies prevented engraftment of JAM-Cpos lymphoma cells in bone marrow, spleen, and lymph nodes of mice. Plasmon resonance studies identified JAM-B as the major ligand for JAM-C, whereas homotypic JAM-C interactions remained at background levels. Accordingly, anti-JAM-C antibodies blocked adhesion of JAM-C-expressing B cells to their ligand JAM-B, and immunofluorescence analysis showed the expression of JAM-B on murine and human lymphatic endothelial cells. Targeting JAM-C could thus constitute a new therapeutic strategy to prevent lymphoma cells from reaching supportive microenvironments not only in the bone marrow and lymph nodes but also in the spleen.

Modulation of the allergen-induced human IgE response in Hu-SCID mice: inhibitory effect of human recombinant IFN-gamma and allergen-derived lipopeptide.

PubMed

Duez, C; Gras-Masse, H; Hammad, H; Akoum, H; Didierlaurent, A; André, C; Tonnel, A B; Pestel, J

2001-01-01

We have previously established a model to study the in vivo human IgE response using humanized SCID mice. Allergic SCID mice were obtained following intraperitoneal injection with mononuclear cells from Dermatophagoides pteronyssinus (Dpt)-sensitive patients, and sensitization by Dpt allergen intraperitoneal injection (immunization) or Dpt aerosol (inhalation). Human serum IgE was measured in allergic SCID mice after administration of human recombinant IFN-gamma or the lipopeptide LP 52-71 (derived from peptide p52-71 from Der p 1, Dpt major allergen, coupled to a lipophilic moiety), during the immunization or the inhalation phase. IFN-gamma inhibited human IgE production when given at the time of immunization, but not during inhalation. This effect was long-lasting as Dpt aerosol, given one month after immunization and IFN-gamma administration, failed to increase IgE levels. Unlike Dpt or p52-71, LP 52-71 failed to induce human IgE production at day 14 and 21 after its injection, but did inhibit the development of the IgE response after a secondary Dpt-challenge. Moreover, LP 52-71 administration 14 days after Dpt inhalation decreased IgE levels, in contrast to peptide 52-71, which increased IgE levels. Thus, taken together these results indicate that the development of the human IgE response in allergic SCID mice can be modulated by modified allergen and a Th1 cytokine.

The Placenta: Applications in Orthopaedic Sports Medicine.

PubMed

McIntyre, James Alexander; Jones, Ian A; Danilkovich, Alla; Vangsness, C Thomas

2018-01-01

Placenta has a long history of use for treating burns and wounds. It is a rich source of collagen and other extracellular matrix proteins, tissue reparative growth factors, and stem cells, including mesenchymal stem cells (MSCs). Recent data show its therapeutic potential for orthopaedic sports medicine indications. To provide orthopaedic surgeons with an anatomic description of the placenta, to characterize its cellular composition, and to review the literature reporting the use of placenta-derived cells and placental tissue allografts for orthopaedic sports medicine indications in animal models and in humans. Systematic review. Using a total of 63 keyword combinations, the PubMed and MEDLINE databases were searched for published articles describing the use of placental cells and/or tissue for orthopaedic sports medicine indications. Information was collected on placental tissue type, indications, animal model, study design, treatment regimen, safety, and efficacy outcomes. Results were categorized by indication and subcategorized by animal model. Outcomes for 29 animal studies and 6 human studies reporting the use of placenta-derived therapeutics were generally positive; however, the placental tissue source, clinical indication, and administration route were highly variable across these studies. Fourteen animal studies described the use of placental tissue for tendon injuries, 13 studies for osteoarthritis or articular cartilage injuries, 3 for ligament injuries, and 1 for synovitis. Both placenta-derived culture-expanded cells (epithelial cells or MSCs) and placental tissue allografts were used in animal studies. In all human studies, commercial placental allografts were used. Five of 6 human studies examined the treatment of foot and ankle pathological conditions, and 1 studied the treatment of knee osteoarthritis. A review of the small number of reported studies revealed a high degree of variability in placental cell types, placental tissue preparation, routes of administration, and treatment regimens, which prohibits making any definitive conclusions. Currently, the clinical use of placenta is limited to only commercial placental tissue allografts, as there are no placenta-derived biological drugs approved for the treatment of orthopaedic sports medicine conditions in the United States. However, this review shows that the application of placental cells or tissue allografts appears to be safe and has potential to improve outcomes for orthopaedic sports medicine indications.

Statins suppress apolipoprotein CIII-induced vascular endothelial cell activation and monocyte adhesion.

PubMed

Zheng, Chunyu; Azcutia, Veronica; Aikawa, Elena; Figueiredo, Jose-Luiz; Croce, Kevin; Sonoki, Hiroyuki; Sacks, Frank M; Luscinskas, Francis W; Aikawa, Masanori

2013-02-01

Activation of vascular endothelial cells (ECs) contributes importantly to inflammation and atherogenesis. We previously reported that apolipoprotein CIII (apoCIII), found abundantly on circulating triglyceride-rich lipoproteins, enhances adhesion of human monocytes to ECs in vitro. Statins may exert lipid-independent anti-inflammatory effects. The present study examined whether statins suppress apoCIII-induced EC activation in vitro and in vivo. Physiologically relevant concentrations of purified human apoCIII enhanced attachment of the monocyte-like cell line THP-1 to human saphenous vein ECs (HSVECs) or human coronary artery ECs (HCAECs) under both static and laminar shear stress conditions. This process mainly depends on vascular cell adhesion molecule-1 (VCAM-1), as a blocking VCAM-1 antibody abolished apoCIII-induced monocyte adhesion. ApoCIII significantly increased VCAM-1 expression in HSVECs and HCAECs. Pre-treatment with statins suppressed apoCIII-induced VCAM-1 expression and monocyte adhesion, with two lipophilic statins (pitavastatin and atorvastatin) exhibiting inhibitory effects at lower concentration than those of hydrophilic pravastatin. Nuclear factor κB (NF-κB) mediated apoCIII-induced VCAM-1 expression, as demonstrated via loss-of-function experiments, and pitavastatin treatment suppressed NF-κB activation. Furthermore, in the aorta of hypercholesterolaemic Ldlr(-/-) mice, pitavastatin administration in vivo suppressed VCAM-1 mRNA and protein, induced by apoCIII bolus injection. Similarly, in a subcutaneous dorsal air pouch mouse model of leucocyte recruitment, apoCIII injection induced F4/80+ monocyte and macrophage accumulation, whereas pitavastatin administration reduced this effect. These findings further establish the direct role of apoCIII in atherogenesis and suggest that anti-inflammatory effects of statins could improve vascular disease in the population with elevated plasma apoCIII.

Prevention and treatment of colon cancer by peroral administration of HAMLET (human α-lactalbumin made lethal to tumour cells).

PubMed

Puthia, Manoj; Storm, Petter; Nadeem, Aftab; Hsiung, Sabrina; Svanborg, Catharina

2014-01-01

Most colon cancers start with dysregulated Wnt/β-catenin signalling and remain a major therapeutic challenge. Examining whether HAMLET (human α-lactalbumin made lethal to tumour cells) may be used for colon cancer treatment is logical, based on the properties of the complex and its biological context. To investigate if HAMLET can be used for colon cancer treatment and prevention. Apc(Min)(/+) mice, which carry mutations relevant to hereditary and sporadic human colorectal tumours, were used as a model for human disease. HAMLET was given perorally in therapeutic and prophylactic regimens. Tumour burden and animal survival of HAMLET-treated and sham-fed mice were compared. Tissue analysis focused on Wnt/β-catenin signalling, proliferation markers and gene expression, using microarrays, immunoblotting, immunohistochemistry and ELISA. Confocal microscopy, reporter assay, immunoprecipitation, immunoblotting, ion flux assays and holographic imaging were used to determine effects on colon cancer cells. Peroral HAMLET administration reduced tumour progression and mortality in Apc(Min)(/+) mice. HAMLET accumulated specifically in tumour tissue, reduced β-catenin and related tumour markers. Gene expression analysis detected inhibition of Wnt signalling and a shift to a more differentiated phenotype. In colon cancer cells with APC mutations, HAMLET altered β-catenin integrity and localisation through an ion channel-dependent pathway, defining a new mechanism for controlling β-catenin signalling. Remarkably, supplying HAMLET to the drinking water from the time of weaning also significantly prevented tumour development. These data identify HAMLET as a new, peroral agent for colon cancer prevention and treatment, especially needed in people carrying APC mutations, where colon cancer remains a leading cause of death.

Prevention and treatment of colon cancer by peroral administration of HAMLET (human α-lactalbumin made lethal to tumour cells)

PubMed Central

Puthia, Manoj; Storm, Petter; Nadeem, Aftab; Hsiung, Sabrina; Svanborg, Catharina

2014-01-01

Background Most colon cancers start with dysregulated Wnt/β-catenin signalling and remain a major therapeutic challenge. Examining whether HAMLET (human α-lactalbumin made lethal to tumour cells) may be used for colon cancer treatment is logical, based on the properties of the complex and its biological context. Objective To investigate if HAMLET can be used for colon cancer treatment and prevention. ApcMin/+ mice, which carry mutations relevant to hereditary and sporadic human colorectal tumours, were used as a model for human disease. Method HAMLET was given perorally in therapeutic and prophylactic regimens. Tumour burden and animal survival of HAMLET-treated and sham-fed mice were compared. Tissue analysis focused on Wnt/β-catenin signalling, proliferation markers and gene expression, using microarrays, immunoblotting, immunohistochemistry and ELISA. Confocal microscopy, reporter assay, immunoprecipitation, immunoblotting, ion flux assays and holographic imaging were used to determine effects on colon cancer cells. Results Peroral HAMLET administration reduced tumour progression and mortality in ApcMin/+ mice. HAMLET accumulated specifically in tumour tissue, reduced β-catenin and related tumour markers. Gene expression analysis detected inhibition of Wnt signalling and a shift to a more differentiated phenotype. In colon cancer cells with APC mutations, HAMLET altered β-catenin integrity and localisation through an ion channel-dependent pathway, defining a new mechanism for controlling β-catenin signalling. Remarkably, supplying HAMLET to the drinking water from the time of weaning also significantly prevented tumour development. Conclusions These data identify HAMLET as a new, peroral agent for colon cancer prevention and treatment, especially needed in people carrying APC mutations, where colon cancer remains a leading cause of death. PMID:23348960

Safety and side effects of cannabidiol, a Cannabis sativa constituent.

PubMed

Bergamaschi, Mateus Machado; Queiroz, Regina Helena Costa; Zuardi, Antonio Waldo; Crippa, José Alexandre S

2011-09-01

Cannabidiol (CBD), a major nonpsychotropic constituent of Cannabis, has multiple pharmacological actions, including anxiolytic, antipsychotic, antiemetic and anti-inflammatory properties. However, little is known about its safety and side effect profile in animals and humans. This review describes in vivo and in vitro reports of CBD administration across a wide range of concentrations, based on reports retrieved from Web of Science, Scielo and Medline. The keywords searched were "cannabinoids", "cannabidiol" and "side effects". Several studies suggest that CBD is non-toxic in non-transformed cells and does not induce changes on food intake, does not induce catalepsy, does not affect physiological parameters (heart rate, blood pressure and body temperature), does not affect gastrointestinal transit and does not alter psychomotor or psychological functions. Also, chronic use and high doses up to 1,500 mg/day of CBD are reportedly well tolerated in humans. Conversely, some studies reported that this cannabinoid can induce some side effects, including inhibition of hepatic drug metabolism, alterations of in vitro cell viability, decreased fertilization capacity, and decreased activities of p-glycoprotein and other drug transporters. Based on recent advances in cannabinoid administration in humans, controlled CBD may be safe in humans and animals. However, further studies are needed to clarify these reported in vitro and in vivo side effects.

Cell-based interventions for neurologic conditions: ethical challenges for early human trials.

PubMed

Mathews, D J H; Sugarman, J; Bok, H; Blass, D M; Coyle, J T; Duggan, P; Finkel, J; Greely, H T; Hillis, A; Hoke, A; Johnson, R; Johnston, M; Kahn, J; Kerr, D; Kurtzberg, J; Liao, S M; McDonald, J W; McKhann, G; Nelson, K B; Rao, M; Regenberg, A; Siegel, A W; Smith, K; Solter, D; Song, H; Vescovi, A; Young, W; Gearhart, J D; Faden, R

2008-07-22

Attempts to translate basic stem cell research into treatments for neurologic diseases and injury are well under way. With a clinical trial for one such treatment approved and in progress in the United States, and additional proposals under review, we must begin to address the ethical issues raised by such early forays into human clinical trials for cell-based interventions for neurologic conditions. An interdisciplinary working group composed of experts in neuroscience, cell biology, bioethics, law, and transplantation, along with leading disease researchers, was convened twice over 2 years to identify and deliberate on the scientific and ethical issues raised by the transition from preclinical to clinical research of cell-based interventions for neurologic conditions. While the relevant ethical issues are in many respects standard challenges of human subjects research, they are heightened in complexity by the novelty of the science, the focus on the CNS, and the political climate in which the science is proceeding. Distinctive challenges confronting US scientists, administrators, institutional review boards, stem cell research oversight committees, and others who will need to make decisions about work involving stem cells and their derivatives and evaluate the ethics of early human trials include evaluating the risks, safety, and benefits of these trials, determining and evaluating cell line provenance, and determining inclusion criteria, informed consent, and the ethics of conducting early human trials in the public spotlight. Further study and deliberation by stakeholders is required to move toward professional and institutional policies and practices governing this research.

Identification of cells initiating human melanomas

PubMed Central

Schatton, Tobias; Murphy, George F.; Frank, Natasha Y.; Yamaura, Kazuhiro; Waaga-Gasser, Ana Maria; Gasser, Martin; Zhan, Qian; Jordan, Stefan; Duncan, Lyn M.; Weishaupt, Carsten; Fuhlbrigge, Robert C.; Kupper, Thomas S.; Sayegh, Mohamed H.; Frank, Markus H.

2012-01-01

Tumour-initiating cells capable of self-renewal and differentiation, which are responsible for tumour growth, have been identified in human haematological malignancies1,2 and solid cancers3–6. If such minority populations are associated with tumour progression in human patients, specific targeting of tumour-initiating cells could be a strategy to eradicate cancers currently resistant to systemic therapy. Here we identify a subpopulation enriched for human malignant-melanoma-initiating cells (MMIC) defined by expression of the chemoresistance mediator ABCB5 (refs 7, 8) and show that specific targeting of this tumorigenic minority population inhibits tumour growth. ABCB5+ tumour cells detected in human melanoma patients show a primitive molecular phenotype and correlate with clinical melanoma progression. In serial human-to-mouse xenotransplantation experiments, ABCB5+ melanoma cells possess greater tumorigenic capacity than ABCB5− bulk populations and re-establish clinical tumour heterogeneity. In vivo genetic lineage tracking demonstrates a specific capacity of ABCB5+ sub-populations for self-renewal and differentiation, because ABCB5+ cancer cells generate both ABCB5+ and ABCB5− progeny, whereas ABCB5− tumour populations give rise, at lower rates, exclusively to ABCB5− cells. In an initial proof-of-principle analysis, designed to test the hypothesis that MMIC are also required for growth of established tumours, systemic administration of a monoclonal antibody directed at ABCB5, shown to be capable of inducing antibody-dependent cell-mediated cytotoxicity in ABCB5+ MMIC, exerted tumour-inhibitory effects. Identification of tumour-initiating cells with enhanced abundance in more advanced disease but susceptibility to specific targeting through a defining chemoresistance determinant has important implications for cancer therapy. PMID:18202660

AFPep: an anti-breast cancer peptide that is orally active.

PubMed

Bennett, James A; DeFreest, Lori; Anaka, Ikenna; Saadati, Hamid; Balulad, Sujata; Jacobson, Herbert I; Andersen, Thomas T

2006-07-01

We have synthesized a cyclic nonapeptide (AFPep) that is effective, after being administered by parenteral routes, for the treatment or the prevention of breast cancer. To test the hypothesis that AFPep remains safe and efficacious after oral administration, three different whole-animal bioassays were utilized, and the mechanism by which AFPep functions was investigated. Using a human breast cancer xenograft model in mice for therapeutic activity, a carcinogen-induced breast cancer model in rats for prevention efficacy, and a mouse uterus growth inhibition model of anti-estrogenic activity, AFPep was administered by oral gavage (p.o.) and its effects compared to those following intraperitoneal (i.p.) and subcutaneous (s.c.) administration. Toxicity studies evaluated body weights and organ weights in mice and rats receiving AFPep. Preliminary mechanistic studies were carried out in T47D human breast cancer cells growing in culture and evaluated the effect of AFPep on estrogen-stimulated cell growth, phosphorylation of the estrogen receptor (ER), and on level of ER-related kinases. Orally administered AFPep stopped the growth of human tumor xenografts in mice, decreased the incidence and multiplicity of breast cancers in carcinogen-exposed rats, and inhibited the estrogen-stimulated growth of mouse uteri. In each of these systems, orally administered AFPep produced an effect similar to that obtained for AFPep administered by either i.p or s.c. routes. In rodents, no evidence of toxicity was seen for the peptide, even at very high doses. In culture, AFPep inhibited the estrogen-stimulated growth, but not the basal growth, of T47D cells, and it inhibited the estrogen-stimulated phosphorylation of Serine 118 in the ER of these cells, which was not explainable by early changes in ER-related kinases. Chronic oral administration of AFPep appears to be safe and effective for the treatment or prevention of breast cancer in animal models.

Interaction of dacarbazine and imexon, in vitro and in vivo, in human A375 melanoma cells.

PubMed

Samulitis, Betty K; Dorr, Robert T; Chow, H-H Sherry

2011-09-01

We evaluated mechanisms of interaction between the alkyating agent dacarbazine (DTIC) and the pro-oxidant, imexon, in the human A375 melanoma cell line. The effect of DTIC and imexon, alone and in combination, was evaluated for growth inhibition (MTT), radiolabeled drug uptake, cellular thiol content (HPLC), and DNA strand breaks (Comet assay). Pharmacokinetic and antitumor effects were evaluated in mice. Growth inhibition in vitro was additive with the two drugs. There was no effect on drug uptake or on the number of DNA strand breaks. There was a >75% reduction in cellular glutathione and cysteine with imexon but not DTIC. Co-administration of the two drugs in mice caused an increase in the area under the curve of both drugs, but the combination was not effective in reducing human A375 melanoma tumors in vivo. Imexon and dacarbazine show additive effects in vitro but not in vivo in human A375 melanoma cells.

Cha-Koji, comprising green tea leaves fermented with Aspergillus luchuensis var kawachii kitahara, increases regulatory T cell production in mice and humans.

PubMed

Yamamoto, Bunsei; Suzuki, Yusuke; Yonezu, Takahisa; Mizushima, Nanami; Watanabe, Nobuo; Sato, Takehito; Inoue, Shigeaki; Inokuchi, Sadaki

2018-03-02

Green tea leaves fermented with Aspergillus luchuensis var kawachii kitahara (Cha-Koji) are a health food containing live A. luchuensis. In this study, we examined the effects of Cha-Koji on the immune system and the enteric environment. First, we designed a clinical trial; after ingesting Cha-Koji daily for 28 days, blood parameters and the fecal composition of the participants were analyzed. Similarly, mice were administered (oral administration) with Cha-Koji suspension or its vehicle for 14 days. Thereafter, both humans and mice were examined by analyzing their immune cell phenotypes and intestinal microbiota. Regulatory T cell (Treg) numbers were significantly increased after administering Cha-Koji. An increase of Clostridium subcluster XIVa, that were known to be rich in butyrate-producing bacterium, was observed in human feces, but not in mice. These results suggest that Cha-Koji has the ability to increase Treg production in both humans and mice, irrespective of the presence of enteric butyrate.

The small-molecule IAP antagonist AT406 inhibits pancreatic cancer cells in vitro and in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang, Yongsheng; Meng, Qinghua; Chen, Bo

In the present study, we tested the anti-pancreatic cancer activity by AT406, a small-molecule antagonist of IAP (inhibitor of apoptosis proteins). In established (Panc-1 and Mia-PaCa-2 lines) and primary human pancreatic cancer cells, treatment of AT406 significantly inhibited cell survival and proliferation. Yet, same AT406 treatment was non-cytotoxic to pancreatic epithelial HPDE6c7 cells. AT406 increased caspase-3/-9 activity and provoked apoptosis in the pancreatic cancer cells. Reversely, AT406′ cytotoxicity in these cells was largely attenuated with pre-treatment of caspase inhibitors. AT406 treatment caused degradation of IAP family proteins (cIAP1 and XIAP) and release of cytochrome C, leaving Bcl-2 unaffected in pancreaticmore » cancer cells. Bcl-2 inhibition (by ABT-737) or shRNA knockdown dramatically sensitized Panc-1 cells to AT406. In vivo, oral administration of AT406 at well-tolerated doses downregulated IAPs (cIAP1/XIAP) and inhibited Panc-1 xenograft tumor growth in severe combined immunodeficient (SCID) nude mice. Together, our preclinical results suggest that AT406 could be further evaluated as a promising anti-pancreatic cancer agent. - Highlights: • AT406 is cytotoxic to established/primary human pancreatic cancer cells. • AT406 provokes caspase-dependent apoptosis in pancreatic cancer cells. • AT406 causes degradation of key IAPs and promotes cytochrome C release. • Bcl-2 inhibition or knockdown dramatically sensitizes Panc-1 cells to AT406. • Oral administration of AT406 inhibits Panc-1 tumor growth in SCID nude mice.« less

HIV-1 infection, response to treatment and establishment of viral latency in a novel humanized T cell-only mouse (TOM) model.

PubMed

Honeycutt, Jenna B; Wahl, Angela; Archin, Nancie; Choudhary, Shailesh; Margolis, David; Garcia, J Victor

2013-10-24

The major targets of HIV infection in humans are CD4⁺ T cells. CD4⁺ T cell depletion is a hallmark of AIDS. Previously, the SCID-hu thy/liv model was used to study the effect of HIV on thymopoeisis in vivo. However, these mice did not develop high levels of peripheral T cell reconstitution and required invasive surgery for infection and analysis. Here, we describe a novel variant of this model in which thy/liv implantation results in systemic reconstitution with human T cells in the absence of any other human hematopoietic lineages. NOD/SCID-hu thy/liv and NSG-hu thy/liv mice were created by implanting human fetal thymus and liver tissues under the kidney capsule of either NOD/SCID or NSG mice. In contrast to NOD/SCID-hu thy/liv mice that show little or no human cells in peripheral blood or tissues, substantial systemic human reconstitution occurs in NSG-hu thy/liv. These mice are exclusively reconstituted with human T cells (i.e. T-cell only mice or TOM). Despite substantial levels of human T cells no signs of graft-versus-host disease (GVHD) were noted in these mice over a period of 14 months. TOM are readily infected after parenteral exposure to HIV-1. HIV replication is sustained in peripheral blood at high levels and results in modest reduction of CD4⁺ T cells. HIV-1 replication in TOM responds to daily administration of combination antiretroviral therapy (ART) resulting in strong suppression of virus replication as determined by undetectable viral load in plasma. Latently HIV infected resting CD4⁺ T cells can be isolated from suppressed mice that can be induced to express HIV ex-vivo upon activation demonstrating the establishment of latency in vivo. NSG-hu thy/liv mice are systemically reconstituted with human T cells. No other human lymphoid lineages are present in these mice (i.e. monocytes/macrophages, B cells and DC are all absent). These T cell only mice do not develop GVHD, are susceptible to HIV-1 infection and can efficiently maintain virus replication. HIV infected TOM undergoing ART harbor latently infected, resting CD4+ T cells.

Oral or parenteral administration of curcumin does not prevent the growth of high-risk t(4;11) acute lymphoblastic leukemia cells engrafted into a NOD/SCID mouse model

USDA-ARS?s Scientific Manuscript database

The efficacy of orally and parenterally administered curcumin was evaluated in NOD.CB17-Prkdcscid/J mice engrafted with the human t(4;11) acute lymphoblastic leukemia line SEM. SEM cells were injected into the tail vein and engraftment was monitored by flow cytometry. Once engraftment was observed...

Analyses of the peripheral immunome following multiple administrations of avelumab, a human IgG1 anti-PD-L1 monoclonal antibody.

PubMed

Donahue, Renee N; Lepone, Lauren M; Grenga, Italia; Jochems, Caroline; Fantini, Massimo; Madan, Ravi A; Heery, Christopher R; Gulley, James L; Schlom, Jeffrey

2017-01-01

Multiple anti-PD-L1/PD-1 checkpoint monoclonal antibodies (MAb) have shown clear evidence of clinical benefit. All except one have been designed or engineered to omit the possibility to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) as a second potential mode of anti-tumor activity; the reason for this is the concern of lysis of PD-L1 positive immune cells. Avelumab is a fully human IgG1 MAb which has been shown in prior in vitro studies to mediate ADCC versus a range of human tumor cells, and clinical studies have demonstrated anti-tumor activity versus a range of human cancers. This study was designed to investigate the effect on immune cell subsets in the peripheral blood of cancer patients prior to and following multiple administrations of avelumab. One hundred twenty-three distinct immune cell subsets in the peripheral blood of cancer patients ( n  = 28) in a phase I trial were analyzed by flow cytometry prior to and following one, three, and nine cycles of avelumab. Changes in soluble (s) CD27 and sCD40L in plasma were also evaluated. In vitro studies were also performed to determine if avelumab would mediate ADCC of PBMC. No statistically significant changes in any of the 123 immune cell subsets analyzed were observed at any dose level, or number of doses, of avelumab. Increases in the ratio of sCD27:sCD40L were observed, suggesting potential immune activation. Controlled in vitro studies also showed lysis of tumor cells by avelumab versus no lysis of PBMC from five donors. These studies demonstrate the lack of any significant effect on multiple immune cell subsets, even those expressing PD-L1, following multiple cycles of avelumab. These results complement prior studies showing anti-tumor effects of avelumab and comparable levels of adverse events with avelumab versus other anti-PD-1/PD-L1 MAbs. These studies provide the rationale to further exploit the potential ADCC mechanism of action of avelumab as well as other human IgG1 checkpoint inhibitors. ClinicalTrials.gov identifier: NCT01772004 (first received: 1/14/13; start date: January 2013) and NCT00001846 (first received date: 11/3/99; start date: August 1999).

Large Gliadin Peptides Detected in the Pancreas of NOD and Healthy Mice following Oral Administration

PubMed Central

Sidenius, Ulrik; Heegaard, Niels H.

2016-01-01

Gluten promotes type 1 diabetes in nonobese diabetic (NOD) mice and likely also in humans. In NOD mice and in non-diabetes-prone mice, it induces inflammation in the pancreatic lymph nodes, suggesting that gluten can initiate inflammation locally. Further, gliadin fragments stimulate insulin secretion from beta cells directly. We hypothesized that gluten fragments may cross the intestinal barrier to be distributed to organs other than the gut. If present in pancreas, gliadin could interact directly with the immune system and the beta cells to initiate diabetes development. We orally and intravenously administered 33-mer and 19-mer gliadin peptide to NOD, BALB/c, and C57BL/6 mice and found that the peptides readily crossed the intestinal barrier in all strains. Several degradation products were found in the pancreas by mass spectroscopy. Notably, the exocrine pancreas incorporated large amounts of radioactive label shortly after administration of the peptides. The study demonstrates that, even in normal animals, large gliadin fragments can reach the pancreas. If applicable to humans, the increased gut permeability in prediabetes and type 1 diabetes patients could expose beta cells directly to gliadin fragments. Here they could initiate inflammation and induce beta cell stress and thus contribute to the development of type 1 diabetes. PMID:27795959

Hippocampal Adult Neurogenesis is Enhanced by Chronic Eszopiclone Treatment in Rats

PubMed Central

Methippara, Melvi; Bashir, Tariq; Suntsova, Natalia; Szymusiak, Ron; McGinty, Dennis

2010-01-01

Summary The adult hippocampal dentate gyrus (DG) exhibits cell proliferation and neurogenesis throughout life. We examined the effects of daily administration of eszopiclone (Esz), a commonly used hypnotic drug and GABA agonist, compared to vehicle, on DG cell proliferation and neurogenesis, and on sleep-wake patterns. Esz was administered during the usual sleep period of rats, to mimic typical use in humans. Esz treatment for 7 days did not affect the rate of cell proliferation, as measured by 5-bromo-2’-deoxyuridine (BrdU) immunostaining. However, twice daily Esz administration for two weeks increased survival of newborn cells, by 46%. Most surviving cells exhibited a neuronal phenotype, identified BrdU-NeuN double-labeling. NeuN (Neuronal nuclei) is a marker of neurons. NREM sleep was increased on day one, but not on days 7 or 14 of Esz administration. Delta EEG activity was increased on days 1 and 7 of treatment, but not on day 14. There is evidence that enhancement of DG neurogenesis is a critical component of the effects of antidepressant treatments of major depressive disorder (MDD). Adult born DG cells are responsive to GABAergic stimulation which promotes cell maturation. The present study suggests that Esz, presumably acting as a GABA agonist, has pro-neurogenic effects in the adult DG. This result is consistent with evidence that Esz enhances antidepressant treatment response of MDD patients with insomnia. PMID:20408925

21 CFR 520.2475 - Toceranib.

Code of Federal Regulations, 2010 CFR

2010-04-01

... Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS ORAL DOSAGE FORM NEW ANIMAL DRUGS § 520.2475 Toceranib. (a) Specifications...) Indications for use. For the treatment of Patnaik grade II or III, recurrent, cutaneous mast cell tumors with...

Constitutive and Treatment-Induced CXCL8-Signalling Selectively Modulates the Efficacy of Anti-Metabolite Therapeutics in Metastatic Prostate Cancer

PubMed Central

Longley, Daniel B.; Wilson, Richard H.; Johnston, Patrick G.; Waugh, David J. J.

2012-01-01

Background The current study was undertaken to characterize the effect of anti-metabolites on inducing CXCL8 signaling and determining whether the constitutive and/or drug-induced CXCL8 signaling in metastatic prostate cancer (CaP) cells modulates their sensitivity to this class of agent. Methods The response of metastatic CaP cells to 5-Fluorouracil (5-FU), Pemetrexed or Tomudex was determined using cell count assays, flow cytometry and PARP cleavage analysis. Quantitative-PCR, ELISA and immunoblots were employed to determine effects of drugs or CXCL8 administration on target gene/protein expression. Results Administration of 5-FU but not pemetrexed potentiated CXCL8 secretion and increased CXCR1 and CXCR2 gene expression in metastatic PC3 cells. Consistent with this, the inhibition of CXCL8 signaling using a CXCR2 antagonist, AZ10397767, increased the cytotoxicity of 5-FU by 4-fold (P<0.001), and increased 5-FU-induced apoptosis in PC3 cells (P<0.01). In contrast, while administration of AZ10397767 had no effect on the sensitivity of pemetrexed, the CXCR2 antagonist exerted the greatest effect in increasing the sensitivity of PC3 cells to Tomudex, a directed thymidylate synthase (TS) inhibitor. Subsequent experiments confirmed that administration of recombinant human CXCL8 increased TS expression, a response mediated in part by the CXCR2 receptor. Moreover, siRNA-mediated knockdown of the CXCL8-target gene Bcl-2 increased the sensitivity of PC3 cells to 5-FU. Conclusions CXCL8 signaling provides a selective resistance of metastatic prostate cancer cells to specific anti-metabolites by promoting a target-associated resistance, in addition to underpinning an evasion of treatment-induced apoptosis. PMID:22590561

Reactivity of retinal blood flow to 100% oxygen breathing after lipopolysaccharide administration in healthy subjects.

PubMed

Kolodjaschna, Julia; Berisha, Fatmire; Lasta, Michael; Polska, Elzbieta; Fuchsjäger-Mayrl, Gabriele; Schmetterer, Leopold

2008-08-01

Administration of low doses of Escherichia coli endotoxin (LPS) to humans enables the study of inflammatory mechanisms. The purpose of the present study was to investigate the retinal vascular reactivity after LPS infusion. In a randomized placebo-controlled cross-over study, 18 healthy male volunteers received 20 IU/kg LPS or placebo as an intravenous bolus infusion. Outcome parameters were measured at baseline and 4h after LPS/placebo administration. At baseline and at 4h after administration a short period of 100% oxygen inhalation was used to assess retinal vasoreactivity to this stimulus. Perimacular white blood cell velocity, density and flux were assessed with the blue-field entoptic technique, retinal branch arterial and venous diameters were measured with a retinal vessel analyzer and red blood cell velocity in retinal branch veins was measured with laser Doppler velocimetry. LPS is associated with peripheral blood leukocytosis and increased white blood cell density in ocular microvessels (p<0.001). In addition, retinal arterial (p=0.02) and venous (p<0.01) diameters were increased. All retinal hemodynamic parameters showed a decrease during 100% oxygen breathing. This decrease was significantly blunted by LPS for all retinal outcome parameters except venous diameter (p=0.04 for white blood cell velocity, p=0.0002 for white blood cell density, p<0.0001 for white blood cell flux, p=0.01 for arterial diameter, p=0.02 for red blood cell velocity and p=0.006 for red blood cell flux). These data indicate that LPS-induced inflammation induces vascular dysregulation in the retina. This may provide a link between inflammation and vascular dysregulation. Further studies are warranted to investigate whether this model may be suitable to study inflammation induced vascular dysregulation in the eye.

Anti-tumor angiogenesis effect of aminopeptidase inhibitor bestatin against B16-BL6 melanoma cells orthotopically implanted into syngeneic mice.

PubMed

Aozuka, Yasushi; Koizumi, Keiichi; Saitoh, Yurika; Ueda, Yasuji; Sakurai, Hiroaki; Saiki, Ikuo

2004-12-08

We investigated the effect of bestatin, an inhibitor of aminopeptidase N (APN)/CD13 and aminopeptidase B, on the angiogenesis induced by B16-BL6 melanoma cells. Oral administration of bestatin (100-200 mg/kg/day) was found to significantly inhibit the melanoma cell-induced angiogenesis in a mouse dorsal air sac assay. Additionally, anti-APN/CD13 mAb (WM15), which neutralizes the aminopeptidase activity in tumor cells, as well as bestatin inhibited the tube-like formation of human umbilical vein endothelial cells (HUVECs) in vitro. Furthermore, the intraperitoneal administration of bestatin (50-100 mg/kg/day) after the orthotopic implantation of B16-BL6 melanoma cells into mice reduced the number of vessels oriented towards the established primary tumor mass on the dorsal side of mice. These findings suggest that bestatin is an active anti-angiogenic agent that may inhibit tumor angiogenesis in vivo and tube-like formation of endothelial cells in vitro through its inhibition of APN/CD13 activity.

Recombinant Lactobacillus plantarum induces immune responses to cancer testis antigen NY-ESO-1 and maturation of dendritic cells

PubMed Central

Mobergslien, Anne; Vasovic, Vlada; Mathiesen, Geir; Fredriksen, Lasse; Westby, Phuong; Eijsink, Vincent GH; Peng, Qian; Sioud, Mouldy

2015-01-01

Given their safe use in humans and inherent adjuvanticity, Lactic Acid Bacteria may offer several advantages over other mucosal delivery strategies for cancer vaccines. The objective of this study is to evaluate the immune responses in mice after oral immunization with Lactobacillus (L) plantarum WCFS1 expressing a cell-wall anchored tumor antigen NY-ESO-1. And to investigate the immunostimulatory potency of this new candidate vaccine on human dendritic cells (DCs). L. plantarum displaying NY-ESO-1 induced NY-ESO-1 specific antibodies and T-cell responses in mice. By contrast, L. plantarum displaying conserved proteins such as heat shock protein-27 and galectin-1, did not induce immunity, suggesting that immune tolerance to self-proteins cannot be broken by oral administration of L. plantarum. With respect to immunomodulation, immature DCs incubated with wild type or L. plantarum-NY-ESO-1 upregulated the expression of co-stimulatory molecules and secreted a large amount of interleukin (IL)-12, TNF-α, but not IL-4. Moreover, they upregulated the expression of immunosuppressive factors such as IL-10 and indoleamine 2,3-dioxygenase. Although L. plantarum-matured DCs expressed inhibitory molecules, they stimulated allogeneic T cells in-vitro. Collectively, the data indicate that L. plantarum-NY-ESO-1 can evoke antigen-specific immunity upon oral administration and induce DC maturation, raising the potential of its use in cancer immunotherapies. PMID:26185907

Fluoxetine Dose and Administration Method Differentially Affect Hippocampal Plasticity in Adult Female Rats

PubMed Central

Pawluski, Jodi L.; van Donkelaar, Eva; Abrams, Zipporah; Steinbusch, Harry W. M.; Charlier, Thierry D.

2014-01-01

Selective serotonin reuptake inhibitor medications are one of the most common treatments for mood disorders. In humans, these medications are taken orally, usually once per day. Unfortunately, administration of antidepressant medications in rodent models is often through injection, oral gavage, or minipump implant, all relatively stressful procedures. The aim of the present study was to investigate how administration of the commonly used SSRI, fluoxetine, via a wafer cookie, compares to fluoxetine administration using an osmotic minipump, with regards to serum drug levels and hippocampal plasticity. For this experiment, adult female Sprague-Dawley rats were divided over the two administration methods: (1) cookie and (2) osmotic minipump and three fluoxetine treatment doses: 0, 5, or 10 mg/kg/day. Results show that a fluoxetine dose of 5 mg/kg/day, but not 10 mg/kg/day, results in comparable serum levels of fluoxetine and its active metabolite norfluoxetine between the two administration methods. Furthermore, minipump administration of fluoxetine resulted in higher levels of cell proliferation in the granule cell layer (GCL) at a 5 mg dose compared to a 10 mg dose. Synaptophysin expression in the GCL, but not CA3, was significantly lower after fluoxetine treatment, regardless of administration method. These data suggest that the administration method and dose of fluoxetine can differentially affect hippocampal plasticity in the adult female rat. PMID:24757568

Interluekin-12 enhances the function and anti-tumor activity in murine and human CD8+ T cells

PubMed Central

Rubinstein, Mark P.; Su, Ee Wern; Suriano, Samantha; Cloud, Colleen A.; Andrijauskaite, Kristina; Kesarwani, Pravin; Schwartz, Kristina M.; Williams, Katelyn; Johnson, C. Bryce; Li, Mingli; Scurti, Gina M.; Salem, Mohamed L.; Paulos, Chrystal M.; Garrett-Mayer, Elizabeth; Mehrotra, Shikhar; Cole, David J.

2016-01-01

Mouse CD8+ T cells conditioned with Interleukin (IL)-12 ex vivo mediate the potent regression of established melanoma when transferred into lymphodepleted mice. However, the quantitative and qualitative changes induced by IL-12 in the responding mouse CD8+ T cells have not been well defined. Moreover, the mechanisms by which IL-12-conditioning impacts human CD8+ T cells, and how such cells might be expanded prior to infusion into patients is not known. We found that ex vivo IL-12-conditioning of mouse CD8+ T cells led to a 10- to 100-fold increase in persistence and anti-tumor efficacy upon adoptive transfer into lymphodepleted mice. The enhancing effect of IL-12 was associated with maintenance of functional avidity. Importantly, in the context of ongoing ACT clinical trials, human CD8+ T cells genetically modified with a tyrosinase-specific T-cell receptor exhibited significantly enhanced functional activity when conditioned with IL-12 as indicated by heightened granzyme B expression and elevated peptide-specific CD107a degranulation. This effect was sustainable despite the 20 days of in vitro cellular expansion required to expand cells over 1,000-fold allowing adequate cell numbers for administration to cancer patients. Overall, these findings support the efficacy and feasibility of ex vivo IL-12-conditioning of TCR-modified human CD8+ T cells for adoptive transfer and cancer therapy. PMID:25676709

Systemic administration of a novel human umbilical cord mesenchymal stem cells population accelerates the resolution of acute liver injury

PubMed Central

2012-01-01

Background Hepatocytes and stem cells transplantation may be an alternative to liver transplantation in acute or chronic liver disease. We aimed to evaluate the therapeutic potential of mesenchymal stem cells from human umbilical cord (UCMSCs), a readily available source of mesenchymal stem cells, in the CCl4-induced acute liver injury model. Methods Mesenchymal stem cells profile was analyzed by flow cytometry. In order to evaluate the capability of our UCMSCs to differentiate in hepatocytes, cells were seeded on three different supports, untreated plastic support, MatrigelTM and human liver acellular matrix. Cells were analyzed by immunocitochemistry for alpha-fetoprotein and albumin expression, qPCR for hepatocyte markers gene expression, Periodic Acid-Schiff staining for glycogen storage, ELISA for albumin detection and colorimetric assay for urea secretion. To assess the effects of undifferentiated UCMSCs in hepatic regeneration after an acute liver injury, we transplanted them via tail vein in mice injected intraperitoneally with a single dose of CCl4. Livers were analyzed by histological evaluation for damage quantification, immunostaining for Kupffer and stellate cells/liver myofibroblasts activation and for UCMSCs homing. Pro- and anti-inflammatory cytokines gene expression was evaluated by qPCR analysis and antioxidant enzyme activity was measured by catalase quantification. Data were analyzed by Mann–Whitney U-test, Kruskal-Wallis test and Cuzick’s test followed by Bonferroni correction for multiple comparisons. Results We have standardized the isolation procedure to obtain a cell population with hepatogenic properties prior to in vivo transplantation. When subjected to hepatogenic differentiation on untreated plastic support, UCMSCs differentiated in hepatocyte-like cells as demonstrated by their morphology, progressive up-regulation of mature hepatocyte markers, glycogen storage, albumin and urea secretion. However, cells seeded on 3D-supports showed a minor or negligible differentiation capacity. UCMSCs-transplanted mice showed a more rapid damage resolution, as shown by histological analysis, with a lower inflammation level and an increased catalase activity compared to CCl4-treated mice. Conclusions Our findings show that UCMSCs can be reliably isolated, have hepatogenic properties and following systemic administration are able to accelerate the resolution of an acute liver injury without any differentiation and manipulation. These features make UCMSCs strong candidates for future application in regenerative medicine for human acute liver disease. PMID:22788801

Suppression of ErbB-2 in androgen-independent human prostate cancer cells enhances cytotoxic effect by gemcitabine in an androgen-reduced environment.

PubMed

Zhang, Li; Davis, Jeffrey S; Zelivianski, Stanislav; Lin, Fen-Fen; Schutte, Rachel; Davis, Thomas L; Hauke, Ralph; Batra, Surinder K; Lin, Ming-Fong

2009-11-18

We examined the efficacy of combination treatments utilizing cytotoxic drugs plus inhibitors to members of the ErbB-ERK signal pathway in human prostate cancer (PCa) LNCaP C-81 cells. Under an androgen-reduced condition, 50nM gemcitabine caused about 40% growth suppression on C-81 cells. Simultaneous treatment of gemcitabine plus 10microM AG825 produced 60% suppression (p<0.03); while, 85% growth inhibition (p<0.02) was seen if AG825 was added to gemcitabine-treated cells after a 24h-interval. Our data thus showed that in androgen-reduced conditions, inhibition of ErbB-2 increases the cytotoxic efficacy of gemcitabine in PCa cells. This finding has significant implications in the choice of drugs for combination therapy as well as the order of administration for treating cancer patients.

Induction of long-lasting multi-specific CD8+ T cells by a four-component DNA-MVA/HIVA-RENTA candidate HIV-1 vaccine in rhesus macaques.

PubMed

Im, Eung-Jun; Nkolola, Joseph P; di Gleria, Kati; McMichael, Andrew J; Hanke, Tomás

2006-10-01

As a part of a long-term effort to develop vaccine against HIV-1 clade A inducing protective T cell responses in humans, we run mutually complementing studies in humans and non-human primates (NHP) with the aim to maximize vaccine immunogenicity. The candidate vaccine under development has four components, pTHr.HIVA and pTH.RENTA DNA, and modified vaccinia virus Ankara (MVA).HIVA and MVA.RENTA, delivered in a heterologous DNA prime-MVA boost regimen. While the HIVA (Gag/epitopes) components have been tested in NHP and over 300 human subjects, we plan to test in humans the RENTA (reverse transcriptase, gp41, Nef, Tat) vaccines designed to broaden HIVA-induced responses in year 2007. Here, we investigated the four-component vaccine long-term immunogenicity in Mamu-A*01-positive rhesus macaques and demonstrated that the vaccine-induced T cells were multi-specific, multi-functional, readily proliferated to recall peptides and were circulating in the peripheral blood of vaccine recipients over 1 year after vaccine administration. The consensus clade A-elicited T cells recognized 50% of tested epitope variants from other HIV-1 clades. Thus, the DNA-MVA/HIVA-RENTA vaccine induced memory T cells of desirable characteristics and similarities to those induced in humans by HIVA vaccines alone; however, single-clade vaccines may not elicit sufficiently cross-reactive responses.

Oral administration of Lactococcus chungangensis inhibits 2,4-dinitrochlorobenzene-induced atopic-like dermatitis in NC/Nga mice.

PubMed

Choi, Woo Jin; Konkit, Maytiya; Kim, Yena; Kim, Mi-Kyung; Kim, Wonyong

2016-09-01

Interest is increasing in the potentially beneficial role of probiotics in the prevention and treatment of atopic diseases. In this study, we investigated the protective effects of Lactococcus chungangensis CAU 28(T) against atopic dermatitis using murine macrophage RAW 264.7 cells, human keratinocyte HaCaT cells, human mast cell line HMC-1 cells, and a 2,4-dinitrochlorobenzene-induced atopic dermatitis model (NC/Nga mice). The results showed that L. chungangensis CAU 28(T) exhibited potent antiinflammatory activity by inhibiting the production of the proinflammatory mediators nitric oxide and prostaglandin E2 in lipopolysaccharide-stimulated RAW 264.7 cells. Treatment with L. chungangensis CAU 28(T) reduced the release of β-hexosaminidase and histamine in HMC-1 cells stimulated with mast cell activator compound 48/80. In addition, the back skin and ears of NC/Nga mice exhibited reduced histological manifestations of atopic skin lesions such as erosion, hyperplasia of the epidermis and dermis, and inflammatory cell infiltration. Oral administration of L. chungangensis CAU 28(T) suppressed the production of IL-4, IL-5, IL-12, IFN-γ, tumor necrosis factor-α, and thymus- and activation-regulated chemokine (TARC) in skin lesions, indicating that it strongly drives the local immune system with efficacy comparable to that of tacrolimus, a topical immunomodulatory drug used for the treatment of atopic dermatitis. The findings indicate that L. chungangensis CAU 28(T) could be a novel probiotic candidate for controlling the symptoms of atopic dermatitis. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Inhibitory effect of maple syrup on the cell growth and invasion of human colorectal cancer cells.

PubMed

Yamamoto, Tetsushi; Uemura, Kentaro; Moriyama, Kaho; Mitamura, Kuniko; Taga, Atsushi

2015-04-01

Maple syrup is a natural sweetener consumed by individuals of all ages throughout the world. Maple syrup contains not only carbohydrates such as sucrose but also various components such as organic acids, amino acids, vitamins and phenolic compounds. Recent studies have shown that these phenolic compounds in maple syrup may possess various activities such as decreasing the blood glucose level and an anticancer effect. In this study, we examined the effect of three types of maple syrup, classified by color, on the cell proliferation, migration and invasion of colorectal cancer (CRC) cells in order to investigate whether the maple syrup is suitable as a phytomedicine for cancer treatment. CRC cells that were administered maple syrup showed significantly lower growth rates than cells that were administered sucrose. In addition, administration of maple syrup to CRC cells caused inhibition of cell invasion, while there was no effect on cell migration. Administration of maple syrup clearly inhibited AKT phosphorylation, while there was no effect on ERK phosphorylation. These data suggest that maple syrup might inhibit cell proliferation and invasion through suppression of AKT activation and be suitable as a phytomedicine for CRC treatment, with fewer adverse effects than traditional chemotherapy.

Inhibitory effect of maple syrup on the cell growth and invasion of human colorectal cancer cells

PubMed Central

YAMAMOTO, TETSUSHI; UEMURA, KENTARO; MORIYAMA, KAHO; MITAMURA, KUNIKO; TAGA, ATSUSHI

2015-01-01

Maple syrup is a natural sweetener consumed by individuals of all ages throughout the world. Maple syrup contains not only carbohydrates such as sucrose but also various components such as organic acids, amino acids, vitamins and phenolic compounds. Recent studies have shown that these phenolic compounds in maple syrup may possess various activities such as decreasing the blood glucose level and an anticancer effect. In this study, we examined the effect of three types of maple syrup, classified by color, on the cell proliferation, migration and invasion of colorectal cancer (CRC) cells in order to investigate whether the maple syrup is suitable as a phytomedicine for cancer treatment. CRC cells that were administered maple syrup showed significantly lower growth rates than cells that were administered sucrose. In addition, administration of maple syrup to CRC cells caused inhibition of cell invasion, while there was no effect on cell migration. Administration of maple syrup clearly inhibited AKT phosphorylation, while there was no effect on ERK phosphorylation. These data suggest that maple syrup might inhibit cell proliferation and invasion through suppression of AKT activation and be suitable as a phytomedicine for CRC treatment, with fewer adverse effects than traditional chemotherapy. PMID:25647359

The nuclear bile acid receptor FXR controls the liver derived tumor suppressor histidine-rich glycoprotein.

PubMed

Deuschle, Ulrich; Birkel, Manfred; Hambruch, Eva; Hornberger, Martin; Kinzel, Olaf; Perović-Ottstadt, Sanja; Schulz, Andreas; Hahn, Ulrike; Burnet, Michael; Kremoser, Claus

2015-06-01

The nuclear bile acid receptor Farnesoid X receptor (FXR) is strongly expressed in liver and intestine, controls bile acid and lipid homeostasis and exerts tumor-protective functions in liver and intestine. Histidine-rich glycoprotein (HRG) is an abundant plasma protein produced by the liver with the proposed function as a pattern recognition molecule involved in the clearance of immune complexes, necrotic cells and pathogens, the modulation of angiogenesis, the normalization of deranged endothelial vessel structure in tumors and tumor suppression. FXR recognition sequences were identified within a human HRG promoter fragment that mediated FXR/FXR-agonist dependent reporter gene activity in vitro. We show that HRG is a novel transcriptional target gene of FXR in human hepatoma cells, human upcyte® primary hepatocytes and 3D human liver microtissues in vitro and in mouse liver in vivo. Prolonged administration of the potent nonsteroidal FXR agonist PX20606 increases HRG levels in mouse plasma. Finally, daily oral administration of this FXR agonist for seven days resulted in a significant increase of HRG levels in the plasma of healthy human male volunteers during a clinical Phase I safety study. HRG might serve as a surrogate marker indicative of liver-specific FXR activation in future human clinical studies. Furthermore, potent FXR agonists might be beneficial in serious health conditions where HRG is reduced, for example, in hepatocellular carcinoma but also other solid cancers, liver failure, sepsis and pre-eclampsia. © 2014 UICC.

Efficient Transplantation via Antibody-based Clearance of Hematopoietic Stem Cell Niches

PubMed Central

Czechowicz, Agnieszka; Kraft, Daniel; Weissman, Irving L.; Bhattacharya, Deepta

2008-01-01

Summary We demonstrate that administration of a depleting antibody specific for c-kit leads to the highly efficient removal of host hematopoietic stem cells (HSCs) and high levels of donor HSC chimerism following transplantation. Upon intravenous transplantation, hematopoietic stem cells (HSCs) can home to specialized niches, yet most HSCs fail to engraft unless recipients are subjected to toxic preconditioning. Here, we provide evidence that, aside from immune barriers, donor HSC engraftment is restricted by occupancy of appropriate niches by host HSCs. Administration of ACK2, an antibody that blocks c-kit function, led to the transient removal of >98% of endogenous HSCs in immunodeficient mice. Subsequent transplantation of these animals with donor HSCs led to chimerism levels of up to 90%. Extrapolation of these methods to humans may enable mild but effective conditioning regimens for transplantation. PMID:18033883

Novel Midkine Inhibitor iMDK Inhibits Tumor Growth and Angiogenesis in Oral Squamous Cell Carcinoma.

PubMed

Masui, Masanori; Okui, Tatsuo; Shimo, Tsuyoshi; Takabatake, Kiyofumi; Fukazawa, Takuya; Matsumoto, Kenichi; Kurio, Naito; Ibaragi, Soichiro; Naomoto, Yoshio; Nagatsuka, Hitoshi; Sasaki, Akira

2016-06-01

Midkine is a heparin-binding growth factor highly expressed in various human malignant tumors. However, its role in the growth of oral squamous cell carcinoma is not well understood. In this study, we analyzed the antitumor effect of a novel midkine inhibitor (iMDK) against oral squamous cell carcinoma. Administration of iMDK induced a robust antitumor response and suppressed cluster of differentiation 31 (CD31) expression in oral squamous cell carcinoma HSC-2 cells and SAS cells xenograft models. iMDK inhibited the proliferation of these cells dose-dependently, as well as the expression of midkine and phospho-extracellular signal-regulated kinase in HSC-2 and SAS cells. Moreover, iMDK significantly inhibited vascular endothelial growth factor and induced tube growth of human umbilical vein endothelial cells in a dose-dependent fashion. These findings suggest that midkine is critically involved in oral squamous cell carcinoma and iMDK can be effectively used for the treatment of oral squamous cell carcinoma. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Specific gene delivery to liver sinusoidal and artery endothelial cells.

PubMed

Abel, Tobias; El Filali, Ebtisam; Waern, Johan; Schneider, Irene C; Yuan, Qinggong; Münch, Robert C; Hick, Meike; Warnecke, Gregor; Madrahimov, Nodir; Kontermann, Roland E; Schüttrumpf, Jörg; Müller, Ulrike C; Seppen, Jurgen; Ott, Michael; Buchholz, Christian J

2013-09-19

Different types of endothelial cells (EC) fulfill distinct tasks depending on their microenvironment. ECs are therefore difficult to genetically manipulate ex vivo for functional studies or gene therapy. We assessed lentiviral vectors (LVs) targeted to the EC surface marker CD105 for in vivo gene delivery. The mouse CD105-specific vector, mCD105-LV, transduced only CD105-positive cells in primary liver cell cultures. Upon systemic injection, strong reporter gene expression was detected in liver where mCD105-LV specifically transduced liver sinusoidal ECs (LSECs) but not Kupffer cells, which were mainly transduced by nontargeted LVs. Tumor ECs were specifically targeted upon intratumoral vector injection. Delivery of the erythropoietin gene with mCD105-LV resulted in substantially increased erythropoietin and hematocrit levels. The human CD105-specific vector (huCD105-LV) transduced exclusively human LSECs in mice transplanted with human liver ECs. Interestingly, when applied at higher dose and in absence of target cells in the liver, huCD105-LV transduced ECs of a human artery transplanted into the descending mouse aorta. The data demonstrate for the first time targeted gene delivery to specialized ECs upon systemic vector administration. This strategy offers novel options to better understand the physiological functions of ECs and to treat genetic diseases such as those affecting blood factors.

Anti-tau antibody administration increases plasma tau in transgenic mice and patients with tauopathy

PubMed Central

Yanamandra, Kiran; Patel, Tirth K.; Jiang, Hong; Schindler, Suzanne; Ulrich, Jason D.; Boxer, Adam L.; Miller, Bruce L.; Kerwin, Diana R.; Gallardo, Gilbert; Stewart, Floy; Finn, Mary Beth; Cairns, Nigel J.; Verghese, Philip B.; Fogelman, Ilana; West, Tim; Braunstein, Joel; Robinson, Grace; Keyser, Jennifer; Roh, Joseph; Knapik, Stephanie S.; Hu, Yan; Holtzman, David M.

2017-01-01

Tauopathies are a group of disorders in which the cytosolic protein tau aggregates and accumulates in cells within the brain, resulting in neurodegeneration. A promising treatment being explored for tauopathies is passive immunization with anti-tau antibodies. We previously found that administration of an anti-tau antibody to human tau transgenic mice increased the concentration of plasma tau. We further explored the effects of administering an anti-tau antibody on plasma tau. After peripheral administration of an anti-tau antibody to human patients with tauopathy and to mice expressing human tau in the central nervous system, there was a dose-dependent increase in plasma tau. In mouse plasma, we found that tau had a short half-life of 8 min that increased to more than 3 hours after administration of anti-tau antibody. As tau transgenic mice accumulated insoluble tau in the brain, brain soluble and interstitial fluid tau decreased. Administration of anti-tau antibody to tau transgenic mice that had decreased brain soluble tau and interstitial fluid tau resulted in an increase in plasma tau, but this increase was less than that observed in tau transgenic mice without these brain changes. Tau transgenic mice subjected to acute neuronal injury using 3-nitropropionic acid showed increased interstitial fluid tau and plasma tau. These data suggest that peripheral administration of an anti-tau antibody results in increased plasma tau, which correlates with the concentration of extracellular and soluble tau in the brain. PMID:28424326

No influence of exogenous hyaluronan on the behavior of human cancer cells or endothelial cell capillary formation.

PubMed

Seino, Satoshi; Takeshita, Fumitaka; Asari, Akira; Masuda, Yasunobu; Kunou, Masaaki; Ochiya, Takahiro

2014-07-01

Hyaluronan (HA), a type of glycosaminoglycan used to construct the extracellular matrix, is involved in the proliferation and motility of cells, including cancer cells. The aim of this study was to determine whether exogenous HA has an influence on cancer in vitro and in vivo. High-molecular-weight HA (900 kDa) and low-molecular-weight HA (10 kDa) were added to several types of cancer cell lines in vitro, and proliferation and invasion were assessed. The effect of HA on capillary formation by human umbilical vein endothelial cells was also analyzed. The results showed that both types of HA had no apparent effect on cellular proliferation, invasion, or capillary formation. In an animal study, the 2 types of HA were orally administered to tumor-bearing mice at a dosage of 200 mg/kg/d for 4 wk. Analysis using an in vivo imaging system revealed that tumor proliferation and metastasis were not greatly altered by HA administration. Furthermore, CD31 immunohistochemical staining revealed no obvious change in tumor microvessels. Taken together, these results demonstrate that exogenously administered HA has little effect on cancer. This study may support the safety of various forms of HA administration, including oral intake. Orally administered hyaluronan was recently found to have beneficial effects. However, the effect of exogenous hyaluronan on cancer remains unclear. Our findings support the safety of orally administered hyaluronan and its use as a functional food ingredient. © 2014 Institute of Food Technologists®

Relaxin attenuates aristolochic acid induced human tubular epithelial cell apoptosis in vitro by activation of the PI3K/Akt signaling pathway.

PubMed

Xie, Xiang-Cheng; Zhao, Ning; Xu, Qun-Hong; Yang, Xiu; Xia, Wen-Kai; Chen, Qi; Wang, Ming; Fei, Xiao

2017-06-01

Aristolochic acid nephropathy remains a leading cause of chronic kidney disease (CKD), however few treatment strategies exist. Emerging evidence has shown that H2 relaxin (RLX) possesses powerful antifibrosis and anti-apoptotic properties, therefore we aimed to investigate whether H2 relaxin can be employed to reduce AA-induced cell apoptosis. Human proximal tubular epithelial (HK-2) cells exposed to AA-I were treated with or without administration of H2 RLX. Cell viability was examined using the WST-8 assay. Apoptotic morphologic alterations were observed using the Hoechst 33342 staining method. Apoptosis was detected using flow cytometry. The expression of caspase 3, caspase 8, caspase 9, ERK1/2, Bax, Bcl-2, and Akt proteins was determined by Western blot. Co-treatment with RLX reversed the increased apoptosis observed in the AA-I only treated group. RLX restored expression of phosphorylated Akt which found to be decreased in the AA-I only treated cells. RLX co-treatment led to a decrease in the Bax/Bcl-2 ratio as well as the cleaved form of caspase-3 compared to the AA-I only treated cells. This anti-apoptotic effect of RLX was attenuated by co-administration of the Akt inhibitor LY294002. The present study demonstrated H2 RLX can decrease AA-I induced apoptosis through activation of the PI3K/Akt signaling pathway.

Improvement of thrombocytopenia following bone marrow transplantation by pegylated recombinant human megakaryocyte growth and development factor in mice.

PubMed

Kabaya, K; Shibuya, K; Torii, Y; Nitta, Y; Ida, M; Akahori, H; Kato, T; Kusaka, M; Miyazaki, H

1996-12-01

We examined whether pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) is capable of improving thrombocytopenia and promoting thrombopoietic reconstitution following lethal irradiation and bone marrow transplantation (BMT) in mice. Immediately after receiving 10 Gy whole body irradiation (day 0), male C3H/HeN mice were inoculated with 10(6) bone marrow cells obtained from syngeneic mice. Circulating platelet counts decreased to below 4% of the normal counts with a nadir on day 10, and then returned to the normal level on day 28 in the control mice undergoing BMT. Subcutaneous consecutive treatment with PEG-rHuMGDF at doses from 10 to 300 micrograms/kg/day from day 1 for 13 days significantly improved the platelet nadir and promoted platelet recovery. The white blood cell counts and hemoglobin concentration following BMT were not influenced by the PEG-rHuMGDF. PEG-rHuMGDF-injection starting from day 5 did not improve the platelet nadir following BMT. Furthermore, administration with PEG-rHuMGDF on alternate days at 55.7 micrograms/kg/day for 7 days or at an interval of 3 days at 78 micrograms/kg/day for 4 days (twice a week for 2 weeks) had a significant efficacy, but these administration regimens had less efficacy than consecutive administration at 30 micrograms/kg/day for 13 days. The numbers of megakaryocytes and megakaryocyte progenitor cells decreased to 5 and 0.2% of normal level, respectively, in the control mice. Consecutive administration of PEG-rHuMGDF enhanced the recovery of the mean number of these cells compared to those in vehicle-treated mice, although such effects were not statistically significant except for the number of megakaryocyte progenitors on day 12. These results suggest that consecutive treatment with PEG-rHuMGDF beginning from the day after BMT may be effective in improving thrombocytopenia following BMT.

Round window administration of gentamicin: a new method for the study of ototoxicity of cochlear hair cells.

PubMed

Husmann, K R; Morgan, A S; Girod, D A; Durham, D

1998-11-01

Damage to inner ear sensory hair cells after systemic administration of ototoxic drugs has been documented in humans and animals. Birds have the ability to regenerate new hair cells to replace those damaged by drugs or noise. Unfortunately, the systemic administration of gentamicin damages both ears in a variable fashion with potentially confounding systemic drug effects. We developed a method of direct application of gentamicin to one cochlea of hatchling chickens, allowing the other ear to serve as a within-animal control. We tested variables including the vehicle for application, location of application, dosage, and duration of gentamicin exposure. After 5 or 28 days survival, the percent length damage to the cochlea and regeneration of hair cells was evaluated using scanning electron microscopy. Controls consisted of the opposite unexposed cochlea and additional animals which received saline instead of gentamicin. Excellent damage was achieved using gentamicin-soaked Gelfoam pledgets applied to the round window membrane. The percent length damage could be varied from 15 to 100% by changing the dosage of gentamicin, with exposures as short as 30 min. No damage was observed in control animals. Regeneration of hair cells was observed in both the base and apex by 28 days survival.

Neural stem cell-based dual suicide gene delivery for metastatic brain tumors.

PubMed

Wang, C; Natsume, A; Lee, H J; Motomura, K; Nishimira, Y; Ohno, M; Ito, M; Kinjo, S; Momota, H; Iwami, K; Ohka, F; Wakabayashi, T; Kim, S U

2012-11-01

In our previous works, we demonstrated that human neural stem cells (NSCs) transduced with the cytosine deaminase (CD) gene showed remarkable 'bystander killer effect' on glioma and medulloblastoma cells after administration of the prodrug 5-fluorocytosine (5-FC). In addition, herpes simplex virus thymidine kinase (TK) is a widely studied enzyme used for suicide gene strategies, for which the prodrug is ganciclovir (GCV). To apply this strategy to brain metastasis treatment, we established here a human NSC line (F3.CD-TK) expressing the dual suicide genes CD and TK. We examined whether F3.CD-TK cells intensified the antitumor effect on lung cancer brain metastases. In vitro studies showed that F3.CD-TK cells exerted a marked bystander effect on human lung cancer cells after treatment with 5-FC and GCV. In a novel experimental brain metastases model, intravenously administered F3 cells migrated near lung cancer metastatic lesions, which were induced by the injection of lung cancer cells via the intracarotid artery. More importantly, F3.CD-TK cells in the presence of prodrugs 5-FC and GCV decreased tumor size and considerably prolonged animal survival. The results of the present study indicate that the dual suicide gene-engineered, NSC-based treatment strategy might offer a new promising therapeutic modality for brain metastases.

21 CFR 1271.350 - Reporting.

Code of Federal Regulations, 2011 CFR

2011-04-01

... UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS Additional Requirements for Establishments Described in § 1271.10... HCT/P involved, and information on all follow-up actions that have been or will be taken in response...

Glycolysis in Panc-1 human pancreatic cancer cells is inhibited by everolimus.

PubMed

Liu, Ling; Gong, Liansheng; Zhang, Yangde; Li, Nianfeng

2013-01-01

The aim of this study was to evaluate the effects and molecular mechanisms of everolimus on Panc-1 human pancreatic cancer cells. Panc-1 human pancreatic cancer cells were treated with everolimus (10 μg/ml) at selected time points (6, 12 and 24 h). Cell proliferation and apoptosis were evaluated by MTT and flow cytometric analyses. The glycolytic activity was determined by measuring the activity of the key enzyme lactate dehydrogenase (LDH) and lactate production. The activity of mammalian target of rapamycin (mTOR) signaling was measured by western blotting. The expression of genes, including hexokinase 2 (HK2) and microRNA-143 (miR-143), was evaluated by real-time polymerase chain reaction (PCR). The administration of everolimus time-dependently inhibited proliferation and glycolysis and induced apoptosis in the Panc-1 human pancreatic cancer cells. As the time of treatment with everolimus increased, the mTOR signaling activity decreased, indicated by lower phosphorylation levels of S6 kinase; however, the phosphorylation levels of mTOR barely changed. Moreover, our data showed an everolimus-induced increase in miR-143 and decrease in HK2 in Panc-1 cells in a time-dependent manner. In conclusion, the current study indicates a novel role of everolimus in its antitumor effect as an inhibitor of glycolysis in Panc-1 human pancreatic cancer cells. Furthermore, our data highlights the significance of exploring the mechanisms of everolimus and miR-143 in malignant tumors.

The Zebrafish Xenograft Platform: Evolution of a Novel Cancer Model and Preclinical Screening Tool.

PubMed

Wertman, Jaime; Veinotte, Chansey J; Dellaire, Graham; Berman, Jason N

2016-01-01

Animal xenografts of human cancers represent a key preclinical tool in the field of cancer research. While mouse xenografts have long been the gold standard, investigators have begun to use zebrafish (Danio rerio) xenotransplantation as a relatively rapid, robust and cost-effective in vivo model of human cancers. There are several important methodological considerations in the design of an informative and efficient zebrafish xenotransplantation experiment. Various transgenic fish strains have been created that facilitate microscopic observation, ranging from the completely transparent casper fish to the Tg(fli1:eGFP) fish that expresses fluorescent GFP protein in its vascular tissue. While human cancer cell lines have been used extensively in zebrafish xenotransplantation studies, several reports have also used primary patient samples as the donor material. The zebrafish is ideally suited for transplanting primary patient material by virtue of the relatively low number of cells required for each embryo (between 50 and 300 cells), the absence of an adaptive immune system in the early zebrafish embryo, and the short experimental timeframe (5-7 days). Following xenotransplantation into the fish, cells can be tracked using in vivo or ex vivo measures of cell proliferation and migration, facilitated by fluorescence or human-specific protein expression. Importantly, assays have been developed that allow for the reliable detection of in vivo human cancer cell growth or inhibition following administration of drugs of interest. The zebrafish xenotransplantation model is a unique and effective tool for the study of cancer cell biology.

PRGF exerts a cytoprotective role in zoledronic acid-treated oral cells.

PubMed

Anitua, Eduardo; Zalduendo, Mar; Troya, María; Orive, Gorka

2016-04-01

Bisphosphonates-related osteonecrosis of the jaw (BRONJ) is a common problem in patients undergoing long-term administration of highly potent nitrogen-containing bisphosphonates (N-BPs). This pathology occurs via bone and soft tissue mechanism. Zoledronic acid (ZA) is the most potent intravenous N-BP used to prevent bone loss in patients with bone dysfunction. The objective of this in vitro study was to evaluate the role of different ZA concentrations on the cells from human oral cavity, as well as the potential of plasma rich in growth factors (PRGF) to overcome the negative effects of this BP. Primary human gingival fibroblasts and primary human alveolar osteoblasts were used. Cell proliferation was evaluated by means of a fluorescence-based method. A colorimetric assay to detect DNA fragmentation undergoing apoptosis was used to determine cell death, and the expression of both NF-κB and pNF-κB were quantified by Western blot analysis. ZA had a cytotoxic effect on both human gingival fibroblasts and human alveolar osteoblasts. This BP inhibits cell proliferation, stimulates apoptosis, and induces inflammation. However, the addition of PRGF suppresses all these negative effects of the ZA. PRGF shows a cytoprotective role against the negative effects of ZA on primary oral cells. At present, there is no definitive treatment for bisphosphonates-related osteonecrosis of the jaw (BRONJ), being mainly palliatives. Our results revealed that PRGF has a cytoprotective role in cells exposed to zoledronic acid, thus providing a reliable adjunctive therapy for the treatment of BRONJ pathology.

Immunotherapy targeting HER2 with genetically modified T cells eliminates tumor-initiating cells in osteosarcoma.

PubMed

Rainusso, N; Brawley, V S; Ghazi, A; Hicks, M J; Gottschalk, S; Rosen, J M; Ahmed, N

2012-03-01

Despite radical surgery and multi-agent chemotherapy, less than one third of patients with recurrent or metastatic osteosarcoma (OS) survive. The limited efficacy of current therapeutic approaches to target tumor-initiating cells (TICs) may explain this dismal outcome. The purpose of this study was to assess the impact of modified T cells expressing a human epidermal growth factor receptor (HER2)-specific chimeric antigen receptor in the OS TIC compartment of human established cell lines. Using the sarcosphere formation assay, we found that OS TICs were resistant to increasing methotrexate concentrations. In contrast, HER2-specific T cells decreased markedly sarcosphere formation capacity and the ability to generate bone tumors in immunodeficient mice after orthotopic transplantation. In vivo, administration of HER2-specific T cells significantly reduced TICs in bulky tumors as judged by decreased sarcosphere forming efficiency in OS cells isolated from explanted tumors. We demonstrate that HER2-specific T cells target drug resistant TICs in established OS cell lines, suggesting that incorporating immunotherapy into current treatment strategies for OS has the potential to improve outcomes.

DMSO Represses Inflammatory Cytokine Production from Human Blood Cells and Reduces Autoimmune Arthritis

PubMed Central

Elisia, Ingrid; Nakamura, Hisae; Lam, Vivian; Hofs, Elyse; Cederberg, Rachel; Cait, Jessica; Hughes, Michael R.; Lee, Leora; Jia, William; Adomat, Hans H.; Guns, Emma S.; McNagny, Kelly M.; Samudio, Ismael; Krystal, Gerald

2016-01-01

Dimethyl sulfoxide (DMSO) is currently used as an alternative treatment for various inflammatory conditions as well as for cancer. Despite its widespread use, there is a paucity of data regarding its safety and efficacy as well as its mechanism of action in human cells. Herein, we demonstrate that DMSO has ex-vivo anti-inflammatory activity using Escherichia coli- (E. coli) and herpes simplex virus-1 (HSV-1)-stimulated whole human blood. Specifically, we found that between 0.5%– 2%, DMSO significantly suppressed the expression of many pro-inflammatory cytokines/chemokines and prostaglandin E2 (PGE2). However, a significant reduction in monocyte viability was also observed at 2% DMSO, suggesting a narrow window of efficacy. Anti-inflammatory concentrations of DMSO suppressed E. coli-induced ERK1/2, p38, JNK and Akt phosphorylation, suggesting DMSO acts on these signaling pathways to suppress inflammatory cytokine/chemokine production. Although DMSO induces the differentiation of B16/F10 melanoma cells in vitro, topical administration of DMSO to mice subcutaneously implanted with B16 melanoma cells was ineffective at reducing tumor growth, DMSO was also found to block mouse macrophages from polarizing to either an M1- or an M2-phenotype, which may contribute to its inability to slow tumor growth. Topical administration of DMSO, however, significantly mitigated K/BxN serum-induced arthritis in mice, and this was associated with reduced levels of pro-inflammatory cytokines in the joints and white blood cell levels in the blood. Thus, while we cannot confirm the efficacy of DMSO as an anti-cancer agent, the use of DMSO in arthritis warrants further investigation to ascertain its therapeutic potential. PMID:27031833

Optimization for the blockade of epidermal growth factor receptor signaling for therapy of human pancreatic carcinoma.

PubMed

Solorzano, C C; Baker, C H; Tsan, R; Traxler, P; Cohen, P; Buchdunger, E; Killion, J J; Fidler, I J

2001-08-01

We determined the optimal administration schedule of a novel epidermal growth factor receptor (EGFR) protein tyrosine kinase inhibitor (PKI), PKI 166 (4-(R)-phenethylamino-6-(hydroxyl)phenyl-7H-pyrrolo[2.3-d]-pyrimidine), alone or in combination with gemcitabine (administered i.p.) for therapy of L3.6pl human pancreatic carcinoma growing in the pancreas of nude mice. Seven days after orthotopic implantation of L3.6pl cells, the mice received daily oral doses of PKI 166. PKI 166 therapy significantly inhibited phosphorylation of the EGFR without affecting EGFR expression. EGFR phosphorylation was restored 72 h after cessation of therapy. Seven days after orthotopic injection of L3.6pl cells, groups of mice received daily or thrice weekly oral doses of PKI 166 alone or in combination with gemcitabine. Treatment with PKI 166 (daily), PKI 166 (3 times/week), or gemcitabine alone produced a 72%, 69%, or 70% reduction in the volume of pancreatic tumors in mice, respectively. Daily oral PKI 166 or thrice weekly oral PKI 166 in combination with injected gemcitabine produced 97% and 95% decreases in volume of pancreatic cancers and significant inhibition of lymph node and liver metastasis. Daily oral PKI 166 produced a 20% decrease in body weight, whereas treatment 3 times/week did not. Decreased microvessel density, decreased proliferating cell nuclear antigen staining, and increased tumor cell and endothelial cell apoptosis correlated with therapeutic success. Collectively, our results demonstrate that three weekly oral administrations of an EGFR tyrosine kinase inhibitor in combination with gemcitabine are sufficient to significantly inhibit primary and metastatic human pancreatic carcinoma.

Multilineage response in aplastic anemia patients following long-term administration of filgrastim (recombinant human granulocyte colony stimulating factor).

PubMed

Sonoda, Y; Ohno, Y; Fujii, H; Takahashi, T; Nakayama, S; Haruyama, H; Nasu, K; Shimazaki, C; Hara, H; Kanamaru, A

1993-11-01

The present multicenter study was undertaken to confirm whether filgrastim/recombinant human granulocyte colony stimulating factor (rhG-CSF) could mobilize residual multipotential stem cells by its G0-shortening effect in patients with aplastic anemia (AA) and induce a multilineage response. Twenty-seven patients with acquired severe or moderate AA received long-term administration (2 to 12+ months) of rhG-CSF in doses from 100 to 400 micrograms/body/day by s.c. injection or 250 to 1,500 micrograms/body/day by i.v. infusion. Twenty-six out of the 27 evaluable patients showed a substantial increase in neutrophils associated with a recovery of myeloid precursors in bone marrow within one month of therapy. Interestingly, 10 out of the 27 patients showed a dramatic improvement in severe anemia after two to ten months of therapy. Moreover, severe thrombocytopenia improved after two to four months of therapy in three out of these ten patients accompanied by a significant increase in megakaryocytes in bone marrow. Clonal cultures of bone marrow cells revealed a recovery in myeloid as well as erythroid precursors in most of these ten patients. In two patients who showed a trilineage response, mixed and megakaryocyte colony formations also recovered. These results suggest that long-term administration of rhG-CSF mobilizes myeloid, erythroid, megakaryocyte and multipotential progenitor cells and induces a multilineage response in some patients with AA.

Involvement of PI3K and ROCK signaling pathways in migration of bone marrow-derived mesenchymal stem cells through human brain microvascular endothelial cell monolayers.

PubMed

Lin, Mei-Na; Shang, De-Shu; Sun, Wei; Li, Bo; Xu, Xin; Fang, Wen-Gang; Zhao, Wei-Dong; Cao, Liu; Chen, Yu-Hua

2013-06-04

Bone marrow-derived mesenchymal stem cells (MSC) represent an important and easily available source of stem cells for potential therapeutic use in neurological diseases. The entry of circulating cells into the central nervous system by intravenous administration requires, firstly, the passage of the cells across the blood-brain barrier (BBB). However, little is known of the details of MSC transmigration across the BBB. In the present study, we employed an in vitro BBB model constructed using a human brain microvascular endothelial cell monolayer to study the mechanism underlying MSC transendothelial migration. Transmigration assays, transendothelial electrical resistance (TEER) and horseradish peroxidase (HRP) flux assays showed that MSC could transmigrate through human brain microvascular endothelial cell monolayers by a paracellular pathway. Cell fractionation and immunofluorescence assays confirmed the disruption of tight junctions. Inhibition assays showed that a Rho-kinase (ROCK) inhibitor (Y27632) effectively promoted MSC transendothelial migration; conversely, a PI3K inhibitor (LY294002) blocked MSC transendothelial migration. Interestingly, adenovirus-mediated interference with ROCK in MSC significantly increased MSC transendothelial migration, and overexpression of a PI3K dominant negative mutant in MSC cells could block transendothelial migration. Our findings provide clear evidence that the PI3K and ROCK pathways are involved in MSC migration through human brain microvascular endothelial cell monolayers. The information yielded by this study may be helpful in constructing gene-modified mesenchymal stem cells that are able to penetrate the BBB effectively for cell therapy. Copyright © 2013 Elsevier B.V. All rights reserved.

Biokinetics of 13C in the human body after oral administration of 13C-labeled glucose as an index for the biokinetics of 14C.

PubMed

Masuda, Tsuyoshi; Tako, Yasuhiro; Matsushita, Kensaku; Takeda, Hiroshi; Endo, Masahiro; Nakamura, Yuji; Hisamatsu, Shun'ichi

2016-09-01

The retention of 13 C in the human body after oral administration of 13 C-labeled glucose was studied in three healthy volunteer subjects to estimate the 50 year cumulative body burden for 13 C as an index of the committed dose of the radioisotope 14 C. After administration of 13 C-labeled glucose, the volunteers ingested controlled diets with a fixed number of calories for 112 d. Samples of breath and urine were collected up to 112 d after administration. Samples of feces were collected up to 14 d after administration. Hair samples were obtained at 119 d after administration and analyzed as a representative index of the rate of excretion of organic 13 C via pathways such as skin cell exfoliation and mucus secretion. All samples were analyzed for 13 C/ 12 C atomic ratio to determine the rate of excretion via each pathway. We then constructed a metabolic model with a total of four pathways (breath, urine, feces, and other) comprising seven compartments. We determined the values of the biokinetic parameters in the model by using the obtained excretion data. From 74% to 94% of the 13 C administered was excreted in breath, whereas  <2% was excreted in urine and feces. In the other pathway, the excretion rate constant in the compartment with the longest residence time stretched to hundreds of days but the rate constant for each subject was not statistically significant (P value  >  0.1). In addition, the dataset for one of the three subjects was markedly different from those of the other two. When we estimated the 50 year cumulative body burden for 13 C by using our model and we included non-statistically significant parameters, a considerable cumulative body burden was found in the compartments excreting to the other pathway. Although our results on the cumulative body burden of 13 C from orally administered carbon as glucose were inconclusive, we found that the compartments excreting to the other pathway had a markedly long residence time and therefore should be studied further to clarify the fate of carbon in the human body. In addition to excreta, data for serum and blood cell samples were also collected from the subjects to examine the metabolism of 13 C in human body.

Generation of recombinant canine interleukin-15 and evaluation of its effects on the proliferation and function of canine NK cells.

PubMed

Lee, Soo-Hyeon; Shin, Dong-Jun; Kim, Sang-Ki

2015-05-15

Interleukin-15 (IL-15) is a pleiotropic cytokine that plays a pivotal role in both innate and adaptive immunity. IL-15 is also a promising cytokine for treating cancer. Despite the growing importance of the clinical use of IL-15 for immunotherapy, no attempts have been made to generate a recombinant canine IL-15 (rcIL-15) and to examine its effects on the antitumor activities of immune effector cells in dogs. Here, we generated an rcIL-15 protein consisting of Asn-49-Ser-162 with a C-terminal His tag and examined its functions ex vivo in terms of the proliferation and antitumor effects on canine non-B, non-T, large granular natural killer (NK) cells. Non-B, non-T, large granular NK cells rapidly expanded in response to stimulation with rcIL-15 in the presence of IL-2, and a majority of the cells that selectively expanded over 21 days exhibited a CD3(-)CD5(-)CD4(-)CD8(+/-)CD21(-) phenotype. Purified rcIL-15 significantly enhanced the expansion rate of canine NK cells derived from peripheral blood mononuclear cells compared to human IL-15, or culture in the absence of IL-15 for 21 days (p<0.05). Purified rcIL-15 was superior at enhancing the effector function of NK cells compared to human IL-15. The cytotoxic activity against canine thyroid adenocarcinoma (CTAC) cells, interferon-γ production, and the mRNA expression levels of perforin and granzyme B of expanded NK cells cultured with rcIL-15 were significantly elevated compared to those cultured with human IL-15 or without IL-15 (p<0.05). Intravenous administration of rcIL-15 significantly increased the numbers of lymphocytes in the peripheral blood of dogs on days 6, 8, and 11 after injection compared to numbers before administration (p<0.05). The results of this study suggest that the rcIL-15 protein, consisting of Asn-49-Ser-162, enhanced the proliferation and antitumor effects of canine NK cells and promoted the generation of lymphocytes in dogs. Copyright © 2015 Elsevier B.V. All rights reserved.

Potential transfer of neurotoxic amino acid β-N-methylamino-alanine (BMAA) from mother to infant during breast-feeding: Predictions from human cell lines.

PubMed

Andersson, Marie; Ersson, Lisa; Brandt, Ingvar; Bergström, Ulrika

2017-04-01

β-N-methylamino-alanine (BMAA) is a non-protein amino acid produced by cyanobacteria, diatoms and dinoflagellates. BMAA has potential to biomagnify in a terrestrial food chain, and to bioaccumulate in fish and shellfish. We have reported that administration of [ 14 C]l-BMAA to lactating mice and rats results in a mother to off-spring transfer via the milk. A preferential enantiomer-specific uptake of [ 14 C]l-BMAA has also been demonstrated in differentiated murine mammary epithelium HC11 cells. These findings, together with neurotoxic effects of BMAA demonstrated both in vitro and in vivo, highlight the need to determine whether such transfer could also occur in humans. Here, we used four cell lines of human origin to examine and compare the transport of the two BMAA enantiomers in vitro. The uptake patterns of [ 14 C]l- and [ 14 C]d-BMAA in the human mammary MCF7 cell line were in agreement with the results in murine HC11 cells, suggesting a potential secretion of BMAA into human breast milk. The permeability coefficients for both [ 14 C]l- and [ 14 C]d-BMAA over monolayers of human intestinal Caco2 cells supported an efficient absorption from the human intestine. As a final step, transport experiments confirmed that [ 14 C]l-and [ 14 C]d-BMAA can be taken up by human SHSY5Y neuroblastoma cells and even more efficiently by human U343 glioblastoma cells. In competition experiments with various amino acids, the ASCT2 specific inhibitor benzylserine was the most effective inhibitor of [ 14 C]l-BMAA uptake tested here. Altogether, our results suggest that BMAA can be transferred from an exposed mother, via the milk, to the brain of the nursed infant. Copyright © 2017. Published by Elsevier Inc.

iNKT-CELL ACTIVATION INDUCES LATE PRETERM BIRTH THAT IS ATTENUATED BY ROSIGLITAZONE1

PubMed Central

St Louis, Derek; Romero, Roberto; Plazyo, Olesya; Arenas-Hernandez, Marcia; Panaitescu, Bogdan; Xu, Yi; Milovic, Tatjana; Xu, Zhonghui; Bhatti, Gaurav; Qing-Sheng, Mi; Drewlo, Sascha; Tarca, Adi L.; Hassan, Sonia S.; Gomez-Lopez, Nardhy

2015-01-01

Preterm birth (PTB) is a leading cause of neonatal morbidity and mortality; however, its non-infection-related mechanisms are poorly understood. Herein, we show that the expansion of activated CD1d-restricted invariant NKT (iNKT) cells in the third trimester by administration of α-galactosylceramide (α-GalCer) induces late PTB and neonatal mortality. In vivo imaging revealed that fetuses from mice that underwent α-GalCer-induced late PTB had bradycardia and died shortly after delivery. Yet, administration of α-GalCer in the second trimester did not cause pregnancy loss. PPARγ activation, through rosiglitazone treatment, reduced the rate of α-GalCer-induced late PTB and improved neonatal survival. Administration of α-GalCer in the third trimester suppressed PPARγ activation as shown by the down-regulation of Fabp4 and Fatp4 in myometrial and decidual tissues, respectively; this suppression was rescued by rosiglitazone treatment. Administration of α-GalCer in the third trimester induced an increase in the activation of conventional CD4+ T cells in myometrial tissues and the infiltration of activated macrophages, neutrophils and mature DCs to myometrial and/or decidual tissues. All of these effects were blunted after rosiglitazone treatment. Administration of α-GalCer also up-regulated the expression of inflammatory genes at the maternal-fetal interface and systemically, and rosiglitazone treatment partially attenuated these responses. Finally, an increased infiltration of activated iNKT-like cells in human decidual tissues is associated with non-infection-related preterm labor/birth. Collectively, these results demonstrate that iNKT-cell activation in vivo leads to late PTB by initiating innate and adaptive immune responses and suggest that the PPARγ pathway has potential as a target for prevention of this syndrome. PMID:26740111

A Phase I Clinical Trial of Systemically Delivered NEMO Binding Domain Peptide in Dogs with Spontaneous Activated B-Cell like Diffuse Large B-Cell Lymphoma

PubMed Central

Habineza Ndikuyeze, Georges; Gaurnier-Hausser, Anita; Patel, Reema; Baldwin, Albert S.; May, Michael J.; Flood, Patrick; Krick, Erika; Propert, Kathleen J.; Mason, Nicola J.

2014-01-01

Activated B-Cell (ABC) Diffuse Large B-Cell Lymphoma (DLBCL) is a common, aggressive and poorly chemoresponsive subtype of DLBCL, characterized by constitutive canonical NF-κB signaling. Inhibition of NF-κB signaling leads to apoptosis of ABC-DLBCL cell lines, suggesting targeted disruption of this pathway may have therapeutic relevance. The selective IKK inhibitor, NEMO Binding Domain (NBD) peptide effectively blocks constitutive NF-κB activity and induces apoptosis in ABC-DLBCL cells in vitro. Here we used a comparative approach to determine the safety and efficacy of systemic NBD peptide to inhibit constitutive NF-κB signaling in privately owned dogs with spontaneous newly diagnosed or relapsed ABC-like DLBCL. Malignant lymph nodes biopsies were taken before and twenty-four hours after peptide administration to determine biological effects. Intravenous administration of <2 mg/kg NBD peptide was safe and inhibited constitutive canonical NF-κB activity in 6/10 dogs. Reductions in mitotic index and Cyclin D expression also occurred in a subset of dogs 24 hours post peptide and in 3 dogs marked, therapeutically beneficial histopathological changes were identified. Mild, grade 1 toxicities were noted in 3 dogs at the time of peptide administration and one dog developed transient subclinical hepatopathy. Long term toxicities were not identified. Pharmacokinetic data suggested rapid uptake of peptide into tissues. No significant hematological or biochemical toxicities were identified. Overall the results from this phase I study indicate that systemic administration of NBD peptide is safe and effectively blocks constitutive NF-κB signaling and reduces malignant B cell proliferation in a subset of dogs with ABC-like DLBCL. These results have potential translational relevance for human ABC-DLBCL. PMID:24798348

Inhibition of human mast cell growth and differentiation by interferon gamma-1b.

PubMed

Kirshenbaum, A S; Worobec, A S; Davis, T A; Goff, J P; Semere, T; Metcalfe, D D

1998-03-01

In an effort to identify cytokines that inhibit human mast cell growth, we cultured HMC-1 cells and recombinant human stem cell factor (rhSCF)-dependent human bone marrow-derived mast cells (HBMCs) in the presence of interferon gamma (IFNgamma)-1b and interferon alpha (IFNalpha)-2b. HMC-1 cell numbers decreased in the presence of 1000 U/mL IFNgamma-1b but were unaffected by 1000 U/mL of IFNalpha-2b. HBMCs were then cultured for 0 to 7 days with 100 ng/mL rhSCF and 10 ng/mL recombinant human IL-3 (rhIL-3), followed by culture in rhSCF and administration of either 1000 U/mL IFNalpha-2b or 1000 U/mL IFNgamma-1b. HBMCs appearing in cultures with rhSCF alone or in combination with IFNalpha-2b were virtually identical in number through 8 weeks of culture. In cultures supplemented with IFNgamma-1b, HBMCs significantly decreased in number and incidence of granular metachromasia by 4 to 5 weeks (p<0.001). Similar results were obtained when human marrow was cultured from day 0 with rhSCF and IFNgamma-1b. Mature rhSCF-dependent HBMCs were also cultured at 5 weeks with rhSCF alone or in combination with IFNgamma-1b. Compared with cells cultured in rhSCF, mature 5-week HBMC cultures treated with rhSCF plus IFNgamma-1b revealed a decrease in mast cells, and those mast cells that remained had fewer toluidine blue- and tryptase-positive granules after 5 to 8 weeks. FACS analysis of rhSCF plus IFNgamma-1b-treated mature HBMCs revealed increased c-kit and Fc(epsilon)RI expression. Mast cell releasibility was not increased. IFNgamma-lb was thus able to suppress mast cell growth from CD34+ cells, suggesting that this agent should be considered as a candidate cytokine for the treatment of disorders of mast cell proliferation.

Clarithromycin expands CD11b+Gr-1+ cells via the STAT3/Bv8 axis to ameliorate lethal endotoxic shock and post-influenza bacterial pneumonia

PubMed Central

Fujii, Hideki; Yagi, Kazuma; Suzuki, Shoji; Hegab, Ahmed E.; Tasaka, Sadatomo; Nakamoto, Nobuhiro; Iwata, Satoshi; Honda, Kenya; Kanai, Takanori; Hasegawa, Naoki; Betsuyaku, Tomoko

2018-01-01

Macrolides are used to treat various inflammatory diseases owing to their immunomodulatory properties; however, little is known about their precise mechanism of action. In this study, we investigated the functional significance of the expansion of myeloid-derived suppressor cell (MDSC)-like CD11b+Gr-1+ cells in response to the macrolide antibiotic clarithromycin (CAM) in mouse models of shock and post-influenza pneumococcal pneumonia as well as in humans. Intraperitoneal administration of CAM markedly expanded splenic and lung CD11b+Gr-1+ cell populations in naïve mice. Notably, CAM pretreatment enhanced survival in a mouse model of lipopolysaccharide (LPS)-induced shock. In addition, adoptive transfer of CAM-treated CD11b+Gr-1+ cells protected mice against LPS-induced lethality via increased IL-10 expression. CAM also improved survival in post-influenza, CAM-resistant pneumococcal pneumonia, with improved lung pathology as well as decreased interferon (IFN)-γ and increased IL-10 levels. Adoptive transfer of CAM-treated CD11b+Gr-1+ cells protected mice from post-influenza pneumococcal pneumonia. Further analysis revealed that the CAM-induced CD11b+Gr-1+ cell expansion was dependent on STAT3-mediated Bv8 production and may be facilitated by the presence of gut commensal microbiota. Lastly, an analysis of peripheral blood obtained from healthy volunteers following oral CAM administration showed a trend toward the expansion of human MDSC-like cells (Lineage−HLA-DR−CD11b+CD33+) with increased arginase 1 mRNA expression. Thus, CAM promoted the expansion of a unique population of immunosuppressive CD11b+Gr-1+ cells essential for the immunomodulatory properties of macrolides. PMID:29621339

Breast milk-derived exosomes promote intestinal epithelial cell growth.

PubMed

Hock, Alison; Miyake, Hiromu; Li, Bo; Lee, Carol; Ermini, Leonardo; Koike, Yuhki; Chen, Yong; Määttänen, Pekka; Zani, Augusto; Pierro, Agostino

2017-05-01

Breast milk administration prevents necrotizing enterocolitis (NEC). However, the mechanism remains unclear. Exosomes are cell-derived vesicles highly present in human milk and regulate intercellular signaling, inflammation, and immune response. We hypothesized that milk-derived exosomes beneficially affect intestinal epithelial cells. Rat milk was collected, and exosomes were isolated using ExoQuick reagent and visualized by Nanoparticle Tracking Analysis. Protein was extracted from encapsulating exosomes, and concentration was measured. 2×10 4 intestinal epithelial cells (IEC-18) were treated for five hours with 0.5-μg/μl exosomes, an equal volume of exosome-free milk, or control solution (PBS). IEC-18 viability was measured using a colorimetric assay (MTT), and gene expression was analyzed by qRT-PCR. Data were compared using one-way ANOVA with Bonferroni post-test. Rat milk was collected, and exosome isolation was confirmed. Compared to control, treatment with exosomes significantly increased IEC viability, proliferation, and stem cell activity (all p<0.05). However, administration of exosome-free milk had less significant effects. Rat milk-derived exosomes promote IEC viability, enhance proliferation, and stimulate intestinal stem cell activity. These findings provide insight into the mechanism of action of breast milk in the intestines. Exosome administration is a promising prevention method for infants at risk of developing NEC when breastfeeding is not tolerated. Copyright © 2017 Elsevier Inc. All rights reserved.

The common γ-chain cytokine IL-7 promotes immunopathogenesis during fungal asthma.

PubMed

Reeder, Kristen M; Dunaway, Chad W; Blackburn, Jonathan P; Yu, Zhihong; Matalon, Sadis; Hastie, Annette T; Ampleford, Elizabeth J; Meyers, Deborah A; Steele, Chad

2018-06-15

Asthmatics sensitized to fungi are reported to have more severe asthma, yet the immunopathogenic pathways contributing to this severity have not been identified. In a pilot assessment of human asthmatics, those subjects sensitized to fungi demonstrated elevated levels of the common γ-chain cytokine IL-7 in lung lavage fluid, which negatively correlated with the lung function measurement PC20. Subsequently, we show that IL-7 administration during experimental fungal asthma worsened lung function and increased the levels of type 2 cytokines (IL-4, IL-5, IL-13), proallergic chemokines (CCL17, CCL22) and proinflammatory cytokines (IL-1α, IL-1β). Intriguingly, IL-7 administration also increased IL-22, which we have previously reported to drive immunopathogenic responses in experimental fungal asthma. Employing IL22 Cre R26R eYFP reporter mice, we identified γδ T cells, iNKT cells, CD4 T cells and ILC3s as sources of IL-22 during fungal asthma; however, only iNKT cells were significantly increased after IL-7 administration. IL-7-induced immunopathogenesis required both type 2 and IL-22 responses. Blockade of IL-7Rα in vivo resulted in attenuated IL-22 production, lower CCL22 levels, decreased iNKT cell, CD4 T-cell and eosinophil recruitment, yet paradoxically increased dynamic lung resistance. Collectively, these results suggest a complex role for IL-7 signaling in allergic fungal asthma.

Allyl isothiocyanate, a constituent of cruciferous vegetables, inhibits growth of PC-3 human prostate cancer xenografts in vivo.

PubMed

Srivastava, Sanjay K; Xiao, Dong; Lew, Karen L; Hershberger, Pamela; Kokkinakis, Demetrius M; Johnson, Candace S; Trump, Donald L; Singh, Shivendra V

2003-10-01

We have shown previously that allyl isothiocyanate (AITC), a constituent of cruciferous vegetables, significantly inhibits survival of PC-3 and LNCaP human prostate cancer cells in culture, whereas proliferation of a normal prostate epithelial cell line is minimally affected by AITC even at concentrations that are highly cytotoxic to the prostate cancer cells. The present studies were designed to test the hypothesis that AITC administration may retard growth of human prostate cancer xenografts in vivo. Bolus i.p. injection of 10 micromol AITC, three times per week (Monday, Wednesday and Friday) beginning the day of tumor cell implantation, significantly inhibited the growth of PC-3 xenograft (P < 0.05 by two-way ANOVA). For example, 26 days after tumor cell implantation, the average tumor volume in control mice (1025 +/- 205 mm3) was approximately 1.7-fold higher compared with AITC-treated mice. Histological analysis of tumors excised at the termination of the experiment revealed a statistically significant increase in number of apoptotic bodies with a concomitant decrease in cells undergoing mitosis in the tumors of AITC-treated mice compared with that of control mice. Western blot analysis indicated an approximately 70% reduction in the levels of anti-apoptotic protein Bcl-2 in the tumor lysate of AITC-treated mice compared with that of control mice. Moreover, the tumors from AITC-treated mice, but not control mice, exhibited cleavage of BID, which is known to promote apoptosis. Statistically significant reduction in the expression of several proteins that regulate G2/M progression, including cyclin B1, cell division cycle (Cdc)25B and Cdc25C (44, 45 and 90% reduction, respectively, compared with control), was also observed in the tumors of AITC-treated mice relative to control tumors. In conclusion, the results of the present study indicate that AITC administration inhibits growth of PC-3 xenografts in vivo by inducing apoptosis and reducing mitotic activity.

Brain transplantation of immortalized human neural stem cells promotes functional recovery in mouse intracerebral hemorrhage stroke model.

PubMed

Lee, Hong J; Kim, Kwang S; Kim, Eun J; Choi, Hyun B; Lee, Kwang H; Park, In H; Ko, Yong; Jeong, Sang W; Kim, Seung U

2007-05-01

We have generated stable, immortalized cell lines of human NSCs from primary human fetal telencephalon cultures via a retroviral vector encoding v-myc. HB1.F3, one of the human NSC lines, expresses a normal human karyotype of 46, XX, and nestin, a cell type-specific marker for NSCs. F3 has the ability to proliferate continuously and differentiate into cells of neuronal and glial lineage. The HB1.F3 human NSC line was used for cell therapy in a mouse model of intracerebral hemorrhage (ICH) stroke. Experimental ICH was induced in adult mice by intrastriatal administration of bacterial collagenase; 1 week after surgery, the rats were randomly divided into two groups so as to receive intracerebrally either human NSCs labeled with beta-galactosidase (n = 31) or phosphate-buffered saline (PBS) (n = 30). Transplanted NSCs were detected by 5-bromo-4-chloro-3-indolyl-beta-d-galactoside histochemistry or double labeling with beta-galactosidase (beta-gal) and mitogen-activated protein (MAP)2, neurofilaments (both for neurons), or glial fibrillary acidic protein (GFAP) (for astrocytes). Behavior of the animals was evaluated for period up to 8 weeks using modified Rotarod tests and a limb placing test. Transplanted human NSCs were identified in the perihematomal areas and differentiated into neurons (beta-gal/MAP2(+) and beta-gal/NF(+)) or astrocytes (beta-gal/GFAP(+)). The NSC-transplanted group showed markedly improved functional performance on the Rotarod test and limb placing after 2-8 weeks compared with the control PBS group (p < .001). These results indicate that the stable immortalized human NSCs are a valuable source of cells for cell replacement and gene transfer for the treatment of ICH and other human neurological disorders. Disclosure of potential conflicts of interest is found at the end of this article.

Armed oncolytic adenovirus expressing PD-L1 mini-body enhances anti-tumor effects of chimeric antigen receptor T-cells in solid tumors

PubMed Central

Tanoue, Kiyonori; Shaw, Amanda Rosewell; Watanabe, Norihiro; Porter, Caroline; Rana, Bhakti; Gottschalk, Stephen; Brenner, Malcolm; Suzuki, Masataka

2017-01-01

Chimeric antigen receptor-modified T cells (CAR T-cells) produce pro-inflammatory cytokines that increase expression of T cell checkpoint signals such as PD-L1, which may inhibit their functionality against solid tumors. In this study, we evaluated in human tumor xenograft models the pro-inflammatory properties of an oncolytic adenovirus (Onc.Ad) with a helper-dependent Ad (HDAd) that expresses a PD-L1 blocking mini-antibody (mini-body) (HDPDL1), as a strategy to enhance CAR T-cell killing. Co-administration of these agents (CAd-VECPDL1) exhibited oncolytic effects with production of PD-L1 mini-body locally at the tumor site. On their own, HDPDL1 exhibited no anti-tumor effect and CAd-VECPDL1 alone reduced tumors only to volumes comparable to Onc.Ad treatment. However, combining CAd-VECPDL1 with HER2.CAR T-cells enhanced anti-tumor activity compared to treatment with either HER2.CAR T-cells alone, or HER2.CAR T-cells plus Onc.Ad. The benefits of locally produced PD-L1 mini-body by CAd-VECPDL1 could not be replicated by infusion of anti-PD-L1 IgG plus HER2.CAR T-cells and co-administration of Onc.Ad in a HER2+ prostate cancer xenograft model. Overall, our data document the superiority of local production of PD-L1 mini-body by CAd-VECPDL1 combined with administration of tumor-directed CAR T-cells to control the growth of solid tumors. PMID:28235763

Development of a new knock-in mouse model and evaluation of pharmacological activities of lusutrombopag, a novel, nonpeptidyl small-molecule agonist of the human thrombopoietin receptor c-Mpl.

PubMed

Yoshida, Hiroshi; Yamada, Hajime; Nogami, Wataru; Dohi, Keiji; Kurino-Yamada, Tomomi; Sugiyama, Koji; Takahashi, Koji; Gahara, Yoshinari; Kitaura, Motoji; Hasegawa, Minoru; Oshima, Itsuki; Kuwabara, Kenji

2018-03-01

Lusutrombopag (S-888711), an oral small-molecule thrombopoietin receptor (TPOR) agonist, has gained first approval as a drug to treat thrombocytopenia of chronic liver disease in patients undergoing elective invasive procedures in Japan. Preclinical studies were performed to evaluate its efficacy against megakaryopoiesis and thrombopoiesis. To investigate the proliferative activity and efficacy of megakaryocytic colony formation via human TPOR, lusutrombopag was applied to cultured human c-Mpl-expressing Ba/F3 (Ba/F3-hMpl) cells and human bone marrow-derived CD34-positive cells, respectively. Lusutrombopag caused a robust increase in Ba/F3-hMpl cells by activating pathways in a manner similar to that of thrombopoietin and induced colony-forming units-megakaryocyte and polyploid megakaryocytes in human CD34-positive cells. Because lusutrombopag has high species specificity for human TPOR, there was no suitable experimental animal model for drug evaluation, except for immunodeficient mouse-based xenograft models. Therefore, a novel genetically modified knock-in mouse, TPOR-Ki/Shi, was developed by replacing mouse Mpl with human-mouse chimera Mpl. In TPOR-Ki/Shi mice, lusutrombopag significantly increased circulating platelets in a dose-dependent manner during 21-day repeated oral administration. Histopathological study of the TPOR-Ki/Shi mice on day 22 also revealed a significant increase in megakaryocytes in the bone marrow. These results indicate that lusutrombopag acts on human TPOR to upregulate differentiation and proliferation of megakaryocytic cells, leading to platelet production. Copyright © 2018 ISEH – Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

Migration of iron-labeled KHYG-1 natural killer cells to subcutaneous tumors in nude mice, as detected by magnetic resonance imaging.

PubMed

Mallett, Christiane L; McFadden, Catherine; Chen, Yuhua; Foster, Paula J

2012-07-01

A novel cell line of cytotoxic natural killer (NK) cells, KHYG-1, was examined in vivo for immunotherapy against prostate cancer. The feasibility of using magnetic resonance imaging (MRI) tracking to monitor the fate of injected NK cells following intravenous (i.v.), intraperitoneal (i.p.) and subcutaneous (s.c.) administration was assessed. PC-3M human prostate cancer cells were injected s.c. into the flank of nude mice (day 0). KHYG-1 NK cells were labeled with an iron oxide contrast agent and injected s.c., i.v. or i.p. on day 8. Mice were imaged by MRI on days 7, 9 and 12. Tumor sections were examined with fluorescence microscopy and immunohistologic staining for NK cells. NK cells were detected in the tumors by histology after all three administration routes. NK cells and fluorescence from the iron label were co-localized. Signal loss was seen in the areas around the tumors and between the tumor lobes in the s.c. group. We are the first to label this cell line of NK cells with an iron oxide contrast agent. Accumulation of NK cells was visualized by MRI after s.c. injection but not after i.v. and i.p. injection.

Suppression of chronic experimental autoimmune neuritis by nasally administered recombinant rat interleukin-6

PubMed Central

DERETZI, G; PELIDOU, S-H; ZOU, L-P; QUIDING, C; MIX, E; LEVI, M; WAHREN, B; ZHU, J

1999-01-01

Experimental autoimmune neuritis (EAN) is a CD4+ T-cell-mediated demyelinating disease of the peripheral nervous system (PNS) and serves as experimental model for human immune-demyelinating neurophathies, especially the Guillain–Barré syndrome. In this study, we examined the effect of recombinant rat interleukin-6 (rrIL-6) on chronic EAN in Lewis rats induced by immunization with P2 peptide 57-81 and Freund’s complete adjuvant (FCA). Nasal administration of rat rIL-6 (1 μg/rat/day) beginning in the initial phase of EAN as a therapeutic agent, decreased the severity and the duration of clinical EAN. Low-grade inflammation and suppression of regional demyelination within the sciatic nerves were seen in rrIL-6-treated rats. Hyporesponsiveness of lymph node T cells, down-regulation of serum tumour necrosis factor-α (TNF-α) and increased levels of P2-specific immunoglobulin G1 (IgG1) antibodies document that nasal administration of rrIL-6 was effective systemically. However, because of the non-specific nature of the treatment and multiple effects of IL-6, more experience and great caution are needed, before nasal administration of IL-6 can be considered as a treatment of human autoimmune demyelinating neurophathies. PMID:10447716

Immunomodulatory effects of ethanol extract of germinated ice plant (Mesembryanthemum crystallinum)

PubMed Central

Choi, Joo-Hee; Jo, Sung-Gang; Jung, Seoung-Ki; Park, Woo-Tae; Kim, Keun-Young; Park, Yong-Wook

2017-01-01

The purpose of this study was to investigate the immunomodulatory activity of ice plant (Mesembryanthemum crystallinum) extract (IPE) in vitro and in vivo. Raji (a human B cell line) and Jurkat (a human T cell line) cells were treated with various doses of IPE and cell proliferation was measured by WST assay. Results showed that IPE promoted the proliferation of both Raji and Jurkat cells in a dose-dependent manner. IPE also enhanced IL-6 and TNF-α production in macrophages in the presence of lipopolysaccharide (LPS), although IPE alone did not induce cytokine production. Moreover, IPE treatment upregulated iNOS gene expression in macrophages in a time- and dose-dependent manner and led to the production of nitric oxide in macrophages in the presence of IFNγ. In vivo studies revealed that oral administration of IPE for 2 weeks increased the differentiation of CD4+, CD8+, and CD19+ cells in splenocytes. These findings suggested that IPE has immunomodulatory effects and could be developed as an immunomodulatory supplement. PMID:28400837

Vinpocetine inhibits breast cancer cells growth in vitro and in vivo.

PubMed

Huang, Er-Wen; Xue, Sheng-Jiang; Zhang, Zheng; Zhou, Jia-Guo; Guan, Yong-Yuan; Tang, Yong-Bo

2012-10-01

Vinpocetine is a clinically used drug for cerebrovascular disorders as well as age-related memory impairment. Of note, vinpocetine has been recently identified as a novel anti-inflammatory agent; however, its effects on cancer cells remain to be investigated. In the present study, we found that vinpocetine potently inhibited proliferation of multiple types of human breast cancer cells by arresting cell cycle at G(0)/G(1) phase. It was also revealed that vinpocetine induced cell apoptosis via mitochondria-dependent pathway. Moreover, vinpocetine impaired the migration of the strongly metastatic cell MDA-MB-231. In xenograft model of human breast cancer in nude mice, both systemic and local administration of vinpocetine significantly suppressed the tumor growth without observed toxicity. Interestingly, vinpocetine markedly attenuated the activation of Akt and signal transducer and activator of transcription factor 3 (STAT3), but had no effects on MAP kinases pathways. Collectively, the data suggest that vinpocetine possesses significant yet previously unknown antitumor properties that may be utilized for the treatment of breast cancer.

21 CFR 862.1785 - Urinary urobilinogen (nonquantitative) test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Urinary urobilinogen (nonquantitative) test system. 862.1785 Section 862.1785 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... urobilinogen (a bile pigment degradation product of red cell hemoglobin) in urine. Estimations obtained by this...

21 CFR 866.5110 - Antiparietal antibody immunological test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... absorbed by the body. The measurements aid in the diagnosis of vitamin B12 deficiency (or pernicious anemia.... 866.5110 Section 866.5110 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... the specific antibody for gastric parietal cells in serum and other body fluids. Gastric parietal...

21 CFR 866.5110 - Antiparietal antibody immunological test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... absorbed by the body. The measurements aid in the diagnosis of vitamin B12 deficiency (or pernicious anemia.... 866.5110 Section 866.5110 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... the specific antibody for gastric parietal cells in serum and other body fluids. Gastric parietal...

21 CFR 866.5110 - Antiparietal antibody immunological test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... absorbed by the body. The measurements aid in the diagnosis of vitamin B12 deficiency (or pernicious anemia.... 866.5110 Section 866.5110 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... the specific antibody for gastric parietal cells in serum and other body fluids. Gastric parietal...

21 CFR 866.5110 - Antiparietal antibody immunological test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... absorbed by the body. The measurements aid in the diagnosis of vitamin B12 deficiency (or pernicious anemia.... 866.5110 Section 866.5110 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... the specific antibody for gastric parietal cells in serum and other body fluids. Gastric parietal...

21 CFR 864.2260 - Chromosome culture kit.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Chromosome culture kit. 864.2260 Section 864.2260 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2260 Chromosome...

21 CFR 640.69 - General requirements.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 7 2010-04-01 2010-04-01 false General requirements. 640.69 Section 640.69 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS... introduce a risk of contamination of the red blood cells and, for plasma intended for injectable products...

21 CFR 864.2260 - Chromosome culture kit.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Chromosome culture kit. 864.2260 Section 864.2260 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2260 Chromosome...

21 CFR 864.2260 - Chromosome culture kit.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Chromosome culture kit. 864.2260 Section 864.2260 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2260 Chromosome...

21 CFR 864.2260 - Chromosome culture kit.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Chromosome culture kit. 864.2260 Section 864.2260 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2260 Chromosome...

21 CFR 864.2260 - Chromosome culture kit.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Chromosome culture kit. 864.2260 Section 864.2260 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2260 Chromosome...

21 CFR 520.2475 - Toceranib.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS... initial dose of 3.25 mg per kilogram (1.48 mg per pound) body weight, orally every other day. (ii) Indications for use. For the treatment of Patnaik grade II or III, recurrent, cutaneous mast cell tumors with...

21 CFR 516.1318 - Masitinib.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS... chapter. (c) Conditions of use in dogs—(1) Amount. 12.5 mg/kilograms (5.7 mg/lb) of body weight daily. (2... mast cell tumors in dogs that have not previously received radiotherapy and/or chemotherapy except...

CD47-blocking immunotherapies stimulate macrophage-mediated destruction of small-cell lung cancer

PubMed Central

Weiskopf, Kipp; Jahchan, Nadine S.; Schnorr, Peter J.; Ring, Aaron M.; Maute, Roy L.; Volkmer, Anne K.; Volkmer, Jens-Peter; Liu, Jie; Lim, Jing Shan; Yang, Dian; Seitz, Garrett; Nguyen, Thuyen; Wu, Di; Guerston, Heather; Trapani, Francesca; George, Julie; Poirier, John T.; Gardner, Eric E.; Miles, Linde A.; de Stanchina, Elisa; Lofgren, Shane M.; Vogel, Hannes; Winslow, Monte M.; Dive, Caroline; Thomas, Roman K.; Rudin, Charles M.; van de Rijn, Matt; Majeti, Ravindra; Garcia, K. Christopher; Weissman, Irving L.

2016-01-01

Small-cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with limited treatment options. CD47 is a cell-surface molecule that promotes immune evasion by engaging signal-regulatory protein alpha (SIRPα), which serves as an inhibitory receptor on macrophages. Here, we found that CD47 is highly expressed on the surface of human SCLC cells; therefore, we investigated CD47-blocking immunotherapies as a potential approach for SCLC treatment. Disruption of the interaction of CD47 with SIRPα using anti-CD47 antibodies induced macrophage-mediated phagocytosis of human SCLC patient cells in culture. In a murine model, administration of CD47-blocking antibodies or targeted inactivation of the Cd47 gene markedly inhibited SCLC tumor growth. Furthermore, using comprehensive antibody arrays, we identified several possible therapeutic targets on the surface of SCLC cells. Antibodies to these targets, including CD56/neural cell adhesion molecule (NCAM), promoted phagocytosis in human SCLC cell lines that was enhanced when combined with CD47-blocking therapies. In light of recent clinical trials for CD47-blocking therapies in cancer treatment, these findings identify disruption of the CD47/SIRPα axis as a potential immunotherapeutic strategy for SCLC. This approach could enable personalized immunotherapeutic regimens in patients with SCLC and other cancers. PMID:27294525

Effect of alterations in metabolic rate on the duration of tolerance in neonatally injected animals.

PubMed

St Rose, J E; Murray, G W; Howe, S A

1976-01-01

Exposure to low environmental temperature caused a decrease in the half-life of human albumin (HA) in rabbits injected with 20 mg HA at birth, and a twofold increase in the proportion of animals which lost their tolerance by 150 days of age. Administration of thyroxin produced an even greater effect with respect to tolerance loss. Simlar mechanisms may be involved in the effects of cold exposure and thyroxin administration on tolerance duration. One possible mecahnism is that the duration of tolerance is dependent upon the metabolic half-life of the tolerance-inducing antigen. An alternative mechanism could be a cold- or thyroxin-induced enhancement of the recruitment of immunologically competent cells from an undifferentiated population of stem cells.

Convection enhanced delivery of panobinostat (LBH589)-loaded pluronic nano-micelles prolongs survival in the F98 rat glioma model.

PubMed

Singleton, W G; Collins, A M; Bienemann, A S; Killick-Cole, C L; Haynes, H R; Asby, D J; Butts, C P; Wyatt, M J; Barua, N U; Gill, S S

2017-01-01

The pan-histone deacetylase inhibitor panobinostat is a potential therapy for malignant glioma, but it is water insoluble and does not cross the blood-brain barrier when administered systemically. In this article, we describe the in vitro and in vivo efficacy of a novel water-soluble nano-micellar formulation of panobinostat designed for administration by convection enhanced delivery (CED). The in vitro efficacy of panobinostat-loaded nano-micelles against rat F98, human U87-MG and M059K glioma cells and against patient-derived glioma stem cells was measured using a cell viability assay. Nano-micelle distribution in rat brain was analyzed following acute CED using rhodamine-labeled nano-micelles, and toxicity was assayed using immunofluorescent microscopy and synaptophysin enzyme-linked immunosorbent assay. We compared the survival of the bioluminescent syngenic F98/Fischer344 rat glioblastoma model treated by acute CED of panobinostat-loaded nano-micelles with that of untreated and vehicle-only-treated controls. Nano-micellar panobinostat is cytotoxic to rat and human glioma cells in vitro in a dose-dependent manner following short-time exposure to drug. Fluorescent rhodamine-labelled nano-micelles distribute with a volume of infusion/volume of distribution (Vi/Vd) ratio of four and five respectively after administration by CED. Administration was not associated with any toxicity when compared to controls. CED of panobinostat-loaded nano-micelles was associated with significantly improved survival when compared to controls (n=8 per group; log-rank test, P <0.001). One hundred percent of treated animals survived the 60-day experimental period and had tumour response on post-mortem histological examination. CED of nano-micellar panobinostat represents a potential novel therapeutic option for malignant glioma and warrants translation into the clinic.

Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 Attenuate Gardnerella vaginalis-Infected Bacterial Vaginosis in Mice

PubMed Central

Jang, Se-Eun; Jeong, Jin-Ju; Choi, Su-Young; Kim, Hyunji; Han, Myung Joo; Kim, Dong-Hyun

2017-01-01

Oral administration of a probiotic mixture (PM; Respecta®) consisting of Lactobacillus rhamnosus HN001 (L1), Lactobacillus acidophilus La-14 (L2), and lactoferrin RCXTM results in colonization of these probiotics in the vagina of healthy women. Therefore, we examined whether vaginal colonization of the PM ingredients L1 and L2 could attenuate bacterial vaginosis (BV). BV was induced in mice via β-estradiol-3-benzoate-induced immunosuppression and intravaginal inoculation with Gardnerella vaginalis (GV). Inflammatory markers were analyzed using enzyme-linked immunosorbent assay, immunoblotting, quantitative polymerase chain reaction, and flow cytometry. Oral or intravaginal administration of PM resulted in colonization of L1 and L2 in the vagina. Oral or intravaginal administration of L1, L2, or PM significantly inhibited GV-induced epithelial cell disruption, myeloperoxidase activity, NF-κB activation, and IL-1β and TNF-α expression (p < 0.05). Administration of these probiotics also inhibited IL-17 and RORγt expression but increased IL-10 and Foxp3 expression. Of these probiotics, L2 most effectively attenuated GV-induced BV, followed by L1 and PM. Oral administration was more effective against GV-induced BV than intravaginal administration. L1 and L2 also significantly inhibited the adherence of GV to HeLa cells (a human cervical cancer cell line) and GV growth in vitro. In addition, L1 and L2 inhibited lipopolysaccharide-induced NF-κB activation in macrophages and the differentiation of splenocytes into Th17 cells in vitro, but increased their differentiation into Treg cells. Our study suggests that L1, L2, and PM attenuated GV-induced vaginosis by regulating both vaginal and systemic innate and adaptive immune responses rather than direct competition or killing of GV in the vagina. PMID:28545241

Nutraceutical augmentation of circulating endothelial progenitor cells and hematopoietic stem cells in human subjects.

PubMed

Mikirova, Nina A; Jackson, James A; Hunninghake, Ron; Kenyon, Julian; Chan, Kyle W H; Swindlehurst, Cathy A; Minev, Boris; Patel, Amit N; Murphy, Michael P; Smith, Leonard; Ramos, Famela; Ichim, Thomas E; Riordan, Neil H

2010-04-08

The medical significance of circulating endothelial or hematopoietic progenitors is becoming increasing recognized. While therapeutic augmentation of circulating progenitor cells using G-CSF has resulted in promising preclinical and early clinical data for several degenerative conditions, this approach is limited by cost and inability to perform chronic administration. Stem-Kine is a food supplement that was previously reported to augment circulating EPC in a pilot study. Here we report a trial in 18 healthy volunteers administered Stem-Kine twice daily for a 2 week period. Significant increases in circulating CD133 and CD34 cells were observed at days 1, 2, 7, and 14 subsequent to initiation of administration, which correlated with increased hematopoietic progenitors as detected by the HALO assay. Augmentation of EPC numbers in circulation was detected by KDR-1/CD34 staining and colony forming assays. These data suggest Stem-Kine supplementation may be useful as a stimulator of reparative processes associated with mobilization of hematopoietic and endothelial progenitors.

HMGB1-stimulated human primary cardiac fibroblasts exert a paracrine action on human and murine cardiac stem cells.

PubMed

Rossini, Alessandra; Zacheo, Antonella; Mocini, David; Totta, Pierangela; Facchiano, Antonio; Castoldi, Raffaella; Sordini, Paolo; Pompilio, Giulio; Abeni, Damiano; Capogrossi, Maurizio C; Germani, Antonia

2008-04-01

High Mobility Box 1 Protein (HMGB1) is a cytokine released into the extracellular space by necrotic cells and activated macrophages in response to injury. We recently demonstrated that HMGB1 administration into the mouse heart during acute myocardial infarction induces cardiac tissue regeneration by activating resident cardiac c-kit+ cells (CSCs) and significantly enhances left ventricular function. In the present study it was analyzed the hypothesis that human cardiac fibroblasts (cFbs) exposed to HMGB1 may exert a paracrine effect on mouse and human CSCs. Human cFbs expressed the HMGB1 receptor RAGE. Luminex technology and ELISA assays revealed that HMGB1 significantly enhanced VEGF, PlGF, Mip-1alpha, IFN-gamma, GM-CSF, Il-10, Il-1beta, Il-4, Il-1ra, Il-9 and TNF-alpha in cFbs cell culture medium. HMGB1-stimulated cFbs conditioned media induced CSC migration and proliferation. These effects were significantly higher to those obtained when HMGB1 was added directly to the culture medium. In conclusion, we provide evidence that HMGB1 may act in a paracrine manner stimulating growth factor, cytokine and chemokine release by cFbs which, in turn, modulate CSC function. Via this mechanism HMGB1 may contribute to cardiac tissue regeneration.

S-allyl derivatives of 6-mercaptopurine are highly potent drugs against human B-CLL through synergism between 6-mercaptopurine and allicin.

PubMed

Miron, Talia; Wilchek, Meir; Shvidel, Lev; Berrebi, Alain; Arditti, Fabian D

2012-12-01

S-allylthio-6-mercaptopurine and its ribose derivative were tested for anti-leukemic activity, using a human- mouse B-CLL model. The novel prodrugs contain two components, a purine analog, which interferes with DNA synthesis, and an S-allylthio, readily engaging in thiol-disulfide exchange reactions. The latter component targets the redox homeostasis which is more sensitive in leukemic cells, than in normal B-cells. Upon administration, the prodrug permeates cells, instantly reacts with free thiol, forming S-allyl mixed disulfides and releasing purine. Several cycles of thiol-disulfide exchange reactions occur, thus extending the duration of the prodrug effects. The concerted action of 2 components, as compared with purine alone, boosted in vitro apoptotis in B-CLL cells from 10% to 38%, and decreased in vivo engraftment of B-CLL from 30% to 0.7%. Copyright © 2012 Elsevier Ltd. All rights reserved.

Three-Dimensional Rotating Wall Vessel-Derived Cell Culture Models for Studying Virus-Host Interactions

PubMed Central

Gardner, Jameson K.; Herbst-Kralovetz, Melissa M.

2016-01-01

The key to better understanding complex virus-host interactions is the utilization of robust three-dimensional (3D) human cell cultures that effectively recapitulate native tissue architecture and model the microenvironment. A lack of physiologically-relevant animal models for many viruses has limited the elucidation of factors that influence viral pathogenesis and of complex host immune mechanisms. Conventional monolayer cell cultures may support viral infection, but are unable to form the tissue structures and complex microenvironments that mimic host physiology and, therefore, limiting their translational utility. The rotating wall vessel (RWV) bioreactor was designed by the National Aeronautics and Space Administration (NASA) to model microgravity and was later found to more accurately reproduce features of human tissue in vivo. Cells grown in RWV bioreactors develop in a low fluid-shear environment, which enables cells to form complex 3D tissue-like aggregates. A wide variety of human tissues (from neuronal to vaginal tissue) have been grown in RWV bioreactors and have been shown to support productive viral infection and physiological meaningful host responses. The in vivo-like characteristics and cellular features of the human 3D RWV-derived aggregates make them ideal model systems to effectively recapitulate pathophysiology and host responses necessary to conduct rigorous basic science, preclinical and translational studies. PMID:27834891

Autophagy mediates cytotoxicity of human colorectal cancer cells treated with garcinielliptone FC.

PubMed

Won, Shen-Jeu; Yen, Cheng-Hsin; Lin, Ting-Yu; Jiang-Shieh, Ya-Fen; Lin, Chun-Nan; Chen, Jyun-Ti; Su, Chun-Li

2018-01-01

The tautomeric pair of garcinielliptone FC (GFC) is a novel tautomeric pair of polyprenyl benzophenonoid isolated from the pericarps of Garcinia subelliptica Merr. (G. subelliptica, Clusiaceae), a tree with abundant sources of polyphenols. Our previous report demonstrated that GFC induced apoptosis on various types of human cancer cell lines including chemoresistant human colorectal cancer HT-29 cells. In the present study, we observed that many autophagy-related genes in GFC-treated HT-29 cells were up- and down-regulated using a cDNA microarray containing oncogenes and kinase genes. GFC-induced autophagy of HT-29 cells was confirmed by observing the formation of acidic vesicular organelles, LC3 puncta, and double-membrane autophagic vesicles using flow cytometry, confocal microscopy, and transmission electron microscopy, respectively. Inhibition of AKT/mTOR/P70S6K signaling as well as formation of Atg5-Atg12 and PI3K/Beclin-1 complexes were observed using Western blot. Administration of autophagy inhibitor (3-methyladenine and shRNA Atg5) and apoptosis inhibitor Z-VAD showed that the GFC-induced autophagy was cytotoxic form and GFC-induced apoptosis enhanced GFC-induced autophagy. Our data suggest the involvement of autophagy and apoptosis in GFC-induced anticancer mechanisms of human colorectal cancer. © 2017 Wiley Periodicals, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhengfu, He; Hu, Zhang; Huiwen, Miao

The search for safe, effective and affordable therapeutics against non-small cell lung cancer (NSCLC) and other lung cancers is important. Here we explored the potential effect of 1-o-acetylbritannilactone (ABL), a novel extract from Inula britannica-F, on angiogenesis and lung cancer cell growth. We demonstrated that ABL dose-dependently inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration, and capillary structure formation of cultured human umbilical vascular endothelial cells (HUVECs). In vivo, ABL administration suppressed VEGF-induced new vasculature formation in Matrigel plugs. For the mechanism investigations, we found that ABL largely inhibited VEGF-mediated activation of Src kinase and focal adhesion kinase (FAK) in HUVECs.more » Furthermore, treatment of A549 NSCLC cells with ABL resulted in cell growth inhibition and Src-FAK in-activation. Significantly, administration of a single dose of ABL (12 mg/kg/day) remarkably suppressed growth of A549 xenografts in nude mice. In vivo microvessels formation and Src activation were also significantly inhibited in ABL-treated xenograft tumors. Taken together, our findings suggest that ABL suppresses angiogenesis and lung cancer cell growth possibly via regulating the VEGFR-Src-FAK signaling. - Highlights: • 1-o-acetylbritannilactone (ABL) inhibits VEGF-induced angiogenesis in vivo. • ABL inhibits VEGF-induced HUVEC migration, proliferation, capillary tube formation. • ABL inhibits VEGF-mediated activation of Src and FAK in HUVECs. • ABL inhibits growth and Src-FAK activation in A549 cells. • ABL administration inhibits A549 tumor angiogenesis and growth in nude mice.« less

IGF-1 receptor targeted nanoparticles for image-guided therapy of stroma-rich and drug resistant human cancer

NASA Astrophysics Data System (ADS)

Zhou, Hongyu; Qian, Weiping; Uckun, Fatih M.; Zhou, Zhiyang; Wang, Liya; Wang, Andrew; Mao, Hui; Yang, Lily

2016-05-01

Low drug delivery efficiency and drug resistance from highly heterogeneous cancer cells and tumor microenvironment represent major challenges in clinical oncology. Growth factor receptor, IGF-1R, is overexpressed in both human tumor cells and tumor associated stromal cells. The level of IGF-1R expression is further up-regulated in drug resistant tumor cells. We have developed IGF-1R targeted magnetic iron oxide nanoparticles (IONPs) carrying multiple anticancer drugs into human tumors. This IGF-1R targeted theranostic nanoparticle delivery system has an iron core for non-invasive MR imaging, amphiphilic polymer coating to ensure the biocompatibility as well as for drug loading and conjugation of recombinant human IGF-1 as targeting molecules. Chemotherapy drugs, Doxorubicin (Dox), was encapsulated into the polymer coating and/or conjugated to the IONP surface by coupling with the carboxyl groups. The ability of IGF1R targeted theranostic nanoparticles to penetrate tumor stromal barrier and enhance tumor cell killing has been demonstrated in human pancreatic cancer patient tissue derived xenograft (PDX) models. Repeated systemic administrations of those IGF-1R targeted theranostic IONP carrying Dox led to breaking the tumor stromal barrier and improved therapeutic effect. Near infrared (NIR) optical and MR imaging enabled noninvasive monitoring of nanoparticle-drug delivery and therapeutic responses. Our results demonstrated that IGF-1R targeted nanoparticles carrying multiple drugs are promising combination therapy approaches for image-guided therapy of stroma-rich and drug resistant human cancer, such as pancreatic cancer.

Shikonin induces apoptosis and inhibits migration of ovarian carcinoma cells by inhibiting the phosphorylation of Src and FAK

PubMed Central

HAO, ZHENFENG; QIAN, JING; YANG, JISHI

2015-01-01

The present study identified that shikonin, a naphthoquinone extracted from the roots of Lithospermum erythrorhizon, inhibits the migration of ovarian cancer cells and induces their apoptosis by impairing the phosphorylation of two kinases, proto-oncogene tyrosine protein kinase Src (Src) and focal adhesion kinase (FAK). Ovarian carcinoma SKOV-3 cells were treated with various concentrations of shikonin and analyzed for the effects on cell migration, invasion and apoptosis via Transwell assays and flow cytometry. In addition, the effects of shikonin administration on the expression and phosphorylation of Src and FAK in the SKOV-3 cells were analyzed by western blotting. Shikonin appeared to induce apoptosis and decrease cell migration in the SKOV-3 ovarian cells. Furthermore, the present study provides evidence that shikonin may exert these effects on human ovarian carcinoma cells via the inhibition of the protein tyrosine kinases, Src and FAK. Thus, shikonin should be considered for additional investigation as a candidate agent for the prevention and treatment of human ovarian cancer. PMID:25621031

Novel pyrimidinic selenourea induces DNA damage, cell cycle arrest, and apoptosis in human breast carcinoma.

PubMed

Barbosa, Flavio A R; Siminski, Tâmila; Canto, Rômulo F S; Almeida, Gabriela M; Mota, Nádia S R S; Ourique, Fabiana; Pedrosa, Rozangela Curi; Braga, Antonio Luiz

2018-06-11

Novel pyrimidinic selenoureas were synthesized and evaluated against tumour and normal cell lines. Among these, the compound named 3j initially showed relevant cytotoxicity and selectivity for tumour cells. Three analogues of 3j were designed and synthesized keeping in view the structural requirements of this compound. Almost all the tested compounds displayed considerable cytotoxicity. However, 8a, one of the 3j analogues, was shown to be highly selective and cytotoxic, especially for breast carcinoma cells (MCF-7) (IC 50  = 3.9 μM). Furthermore, 8a caused DNA damage, inhibited cell proliferation, was able to arrest cell cycle in S phase, and induced cell death by apoptosis in human breast carcinoma cells. Moreover, predictions of pharmacokinetic properties showed that 8a may present good absorption and permeation characteristics for oral administration. Overall, the current study established 8a as a potential drug prototype to be employed as a DNA interactive cytotoxic agent for the treatment of breast cancer. Copyright © 2018. Published by Elsevier Masson SAS.

A Novel Peptide to Treat Oral Mucositis Blocks Endothelial and Epithelial Cell Apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu Xiaoyan; Chen Peili; Sonis, Stephen T.

2012-07-01

Purpose: No effective agents currently exist to treat oral mucositis (OM) in patients receiving chemoradiation for the treatment of head-and-neck cancer. We identified a novel 21-amino acid peptide derived from antrum mucosal protein-18 that is cytoprotective, mitogenic, and motogenic in tissue culture and animal models of gastrointestinal epithelial cell injury. We examined whether administration of antrum mucosal protein peptide (AMP-p) could protect against and/or speed recovery from OM. Methods and Materials: OM was induced in established hamster models by a single dose of radiation, fractionated radiation, or fractionated radiation together with cisplatin to simulate conventional treatments of head-and-neck cancer. Results:more » Daily subcutaneous administration of AMP-p reduced the occurrence of ulceration and accelerated mucosal recovery in all three models. A delay in the onset of erythema after irradiation was observed, suggesting that a protective effect exists even before injury to mucosal epithelial cells occurs. To test this hypothesis, the effects of AMP-p on tumor necrosis factor-{alpha}-induced apoptosis were studied in an endothelial cell line (human dermal microvascular endothelial cells) as well as an epithelial cell line (human adult low-calcium, high-temperature keratinocytes; HaCaT) used to model the oral mucosa. AMP-p treatment, either before or after cell monolayers were exposed to tumor necrosis factor-{alpha}, protected against development of apoptosis in both cell types when assessed by annexin V and propidium iodide staining followed by flow cytometry or ligase-mediated polymerase chain reaction. Conclusions: These observations suggest that the ability of AMP-p to attenuate radiation-induced OM could be attributable, at least in part, to its antiapoptotic activity.« less

Synergistic Inhibition of Thalidomide and Icotinib on Human Non-Small Cell Lung Carcinomas Through ERK and AKT Signaling.

PubMed

Sun, Xiang; Xu, Yang; Wang, Yi; Chen, Qian; Liu, Liu; Bao, Yangyi

2018-05-15

BACKGROUND Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have been widely used in the treatment of non-small cell lung cancer (NSCLC) patients with sensitive EGFR mutations. However, the survival of patients with EGFR-TKI administration is limited by the inevitable development of acquired drug resistance. Recently, multi-targeted drugs combination has been shown to be a promising strategy to improve the efficacy of EGFR-TKI treatment and enable the reduction of drug resistance in NSCLC. MATERIAL AND METHODS Humanized NSCLC cell lines PC9 and A549 were co-cultured with thalidomide and/or icotinib to test for anti-tumor efficiency. Cell proliferation was measured by MTT assay, cell apoptosis by flow cytometry and cell migration by wound healing assay. Western blot was performed to determine the expression of caspase-3, -8, -9, Bax, EGFR, VEGF-R, AKT, ERK, MMP2, MMP9, and NF-κB. The xenograft mouse model was used to explore the effects of thalidomide and icotinib in vivo. Immunohistochemical testing was used to determine the expression of Ki-67 and TUNEL staining in tumor tissues. RESULTS Treatments of thalidomide and/or icotinib reduced cell viability, induced apoptosis, and suppressed migration. Attenuation of pEGFR and pVEGF-R resulted in deactivation of ERK and AKT pathways, which eventually increased the anti-proliferative response. In PC9 xenograft model, combined administration of thalidomide and icotinib restrained tumor growth with remarkable reduced Ki-67 index and increased TUNEL positive cells. CONCLUSIONS Thalidomide sensitizes icotinib to increase apoptosis and prevent migration, and it may be a potentially promising anti-tumor drug in lung cancer multi-modality therapy.

Activated glycoprotein A repetitions predominant (GARP)-expressing regulatory T cells inhibit allergen-induced intestinal inflammation in humanized mice.

PubMed

Eschborn, Melanie; Weigmann, Benno; Reissig, Sonja; Waisman, Ari; Saloga, Joachim; Bellinghausen, Iris

2015-07-01

Recently, we developed a humanized mouse model of allergen-induced IgE-dependent gut inflammation in PBMC-engrafted immunodeficient mice. In the present study, we wanted to investigate the role of regulatory T (Treg) cells and their activation status in this model. Nonobese diabetic-severe combined immunodeficiency-γc(-/-) mice were injected intraperitoneally with human PBMCs from allergic donors together with the respective allergen or NaCl as control in the presence or absence of different concentrations of CD4(+)CD25(+) Treg cells of the same donor. After an additional allergen boost 1 week later, mice were challenged with the allergen rectally on day 21 and gut inflammation was monitored by a high-resolution video mini-endoscopic system evaluating translucency, granularity, fibrin production, vascularity, and stool. Allergen-specific human IgE in mouse sera, which was detectable only in PBMC plus allergen-treated mice, was strongly inhibited by coinjection of Treg cells at a ratio of at least 1:10. Consequently, the presence of Treg cells significantly decreased IgE-dependent allergen-induced gut inflammation after rectal allergen challenge. In addition, Treg cells reduced allergen-specific proliferation and cytokine production of recovered human CD4(+) T cells in vitro. Activation of Treg cells before injection further increased all inhibitory effects. Prevention of gut inflammation also occurred by the administration of glycoprotein A repetitions predominant, a molecule expressed by activated Treg cells, whereas its blockade completely abrogated inhibition by Treg cells. These results demonstrate that allergen-specific gut inflammation in human PBMC-engrafted mice can be avoided by enhancing the numbers or activity of autologous Treg cells, which is of great interest for therapeutic intervention of allergic diseases of the intestine. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Comparative photodynamic therapy cytotoxicity of mannose-conjugated chlorin and talaporfin sodium in cultured human and rat cells.

PubMed

Shinoda, Yo; Takahashi, Tsutomu; Akimoto, Jiro; Ichikawa, Megumi; Yamazaki, Hiromi; Narumi, Atsushi; Yano, Shigenobu; Fujiwara, Yasuyuki

2017-01-01

Photodynamic therapy (PDT) is a Food and Drug Administration authorized method for cancer treatment, which uses photosensitizer and laser photo-irradiation to generate reactive oxygen species to induce cell death in tumors. Photosensitizers have been progressively developed, from first to third generation, with improvements in cell specificity, reduced side effects and toxicity, increased sensitivity for irradiation and reduced persistence of photosensitizer in healthy cells. These improvements have been achieved by basic comparative experiments between current and novel photosensitizers using cell lines; however, photosensitizers should be carefully evaluated because they may have cell type specificity. In the present study, we compared a third-generation photosensitizer, β-mannose-conjugated chlorin (β-M-chlorin), with the second generation, talaporfin sodium (NPe6), using seven different rat and human cell lines and a neuronal/glial primary culture prepared from rat embryos. NPe6 was more effective than β-M-chlorin in human-derived cell lines, and β-M-chlorin was more effective than NPe6 in rat primary cultures and rat-derived cell lines, except for the rat pheochromocytoma cell line, PC12. These differences of phototoxicity in different cell types are not because of differences in photosensitivity between the photosensitizers, but rather are associated with different distribution and accumulation rates in the different cell types. These data suggest that evaluation of photosensitizers for PDT should be carried out using as large a variety of cell types as possible because each photosensitizer may have cell type specificity.

Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Reveal Bradycardiac Effects Caused by Co-Administration of Sofosbuvir and Amiodarone.

PubMed

Yu, Yankun; Liu, Feng; He, Liuming; Ramakrishna, Seeram; Zheng, Monica; Bu, Lei; Xu, Ying

2018-05-30

Co-administration of sofosbuvir, an anti-hepatitis C virus medication, and antiarrhythmic amiodarone causes symptomatic severe bradycardia in patients and animal models. However, in a few in vitro studies, the combination of sofosbuvir and amiodarone resulted in tachycardiac effects in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). This discrepancy may be attributable to the use of immature hiPSC-CMs in the in vitro studies. To address this, we evaluated the ability of our in-house hiPSC-CMs to assess the interactions between sofosbuvir and amiodarone in vitro. We performed whole-cell patch recordings on hiPSC-CMs to examine the cardiac effect of sofosbuvir and amiodarone, alone or in combination. We found that sofosbuvir and amiodarone caused bradycardiac effects (the beating rate decreased to 75% of the vehicle control, P < 0.001) on our hiPSC-CMs when applied in combination, but they had no significant effect when applied alone. Furthermore, the bradycardiac effect was membrane potential dependent: it increased with depolarization. This raised the possibility that the bradycardiac effects in vivo may originate in nodal cells, which have a more depolarized resting membrane potential compared with ventricular cells. The bradycardiac effects of sofosbuvir plus amiodarone in vitro are consistent with the clinical phenotype and suggest that our hiPSC-CMs may serve as a useful tool in assessing cardiac safety during drug discovery and development process.

An Abundant Perivascular Source of Stem Cells for Bone Tissue Engineering

PubMed Central

James, Aaron W.; Zara, Janette N.; Corselli, Mirko; Askarinam, Asal; Zhou, Ann M.; Hourfar, Alireza; Nguyen, Alan; Megerdichian, Silva; Asatrian, Greg; Pang, Shen; Stoker, David; Zhang, Xinli; Wu, Benjamin

2012-01-01

Adipose tissue is an ideal mesenchymal stem cell (MSC) source, as it is dispensable and accessible with minimal morbidity. However, the stromal vascular fraction (SVF) of adipose tissue is a heterogeneous cell population, which has disadvantages for tissue regeneration. In the present study, we prospectively purified human perivascular stem cells (PSCs) from n = 60 samples of human lipoaspirate and documented their frequency, viability, and variation with patient demographics. PSCs are a fluorescence-activated cell sorting-sorted population composed of pericytes (CD45−, CD146+, CD34−) and adventitial cells (CD45−, CD146−, CD34+), each of which we have previously reported to have properties of MSCs. Here, we found that PSCs make up, on average, 43.2% of SVF from human lipoaspirate (19.5% pericytes and 23.8% adventitial cells). These numbers were minimally changed by age, gender, or body mass index of the patient or by length of refrigerated storage time between liposuction and processing. In a previous publication, we observed that human PSCs (hPSCs) formed significantly more bone in vivo in comparison with unsorted human SVF (hSVF) in an intramuscular implantation model. We now extend this finding to a bone injury model, observing that purified hPSCs led to significantly greater healing of mouse critical-size calvarial defects than hSVF (60.9% healing as opposed to 15.4% healing at 2 weeks postoperative by microcomputed tomography analysis). These studies suggest that adipose-derived hPSCs are a new cell source for future efforts in skeletal regenerative medicine. Moreover, hPSCs are a stem cell-based therapeutic that is readily approvable by the U.S. Food and Drug Administration, with potentially increased safety, purity, identity, potency, and efficacy. PMID:23197874

Identification and characterization of enhancer agonist human cytotoxic T-cell epitopes of the human papillomavirus type 16 (HPV16) E6/E7

PubMed Central

Tsang, Kwong Y.; Fantini, Massimo; Fernando, Romaine I.; Palena, Claudia; David, Justin M.; Hodge, James W.; Gabitzsch, Elizabeth S.; Jones, Frank R.; Schlom, Jeffrey

2017-01-01

Human papillomavirus (HPV) is associated with the etiology of cervical carcinoma, head and neck squamous cell carcinoma, and several other cancer types. Vaccines directed against HPV virus-like particles and coat proteins have been extremely successful in the prevention of cervical cancer through the activation of host HPV-specific antibody responses; however, HPV-associated cancers remain a major public health problem. The development of a therapeutic vaccine will require the generation of T-cell responses directed against early HPV proteins (E6/E7) expressed in HPV-infected tumor cells. Clinical studies using various vaccine platforms have demonstrated that both HPV-specific human T cells can be generated and patient benefit can be achieved. However, no HPV therapeutic vaccine has been approved by the Food and Drug Administration to date. One method of enhancing the potential efficacy of a therapeutic vaccine is the generation of agonist epitopes. We report the first description of enhancer cytotoxic T lymphocyte agonist epitopes for HPV E6 and E7. While the in silico algorithm revealed six epitopes with potentially improved binding to human leukocyte antigen–A2 allele (HLA-A2)–Class I, 5/6 demonstrated enhanced binding to HLA-Class I in cell-based assays and only 3/6 had a greater ability to activate HPV-specific T cells which could lyse tumor cells expressing native HPV, compared to their native epitope counterparts. These agonist epitopes have potential for use in a range of HPV therapeutic vaccine platforms and for use in HPV-specific adoptive T- or natural killer–cell platforms. PMID:28389098

Human platelet antigen (HPA)-1a peptides do not reliably suppress anti-HPA-1a responses using a humanized severe combined immunodeficiency (SCID) mouse model

PubMed Central

Jackson, D J; Eastlake, J L; Kumpel, B M

2014-01-01

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) occurs most frequently when human platelet antigen (HPA)-1a-positive fetal platelets are destroyed by maternal HPA-1a immunoglobulin (Ig)G antibodies. Pregnancies at risk are treated by administration of high-dose intravenous Ig (IVIG) to women, but this is expensive and often not well tolerated. Peptide immunotherapy may be effective for ameliorating some allergic and autoimmune diseases. The HPA-1a/1b polymorphism is Leu/Pro33 on β3 integrin (CD61), and the anti-HPA-1a response is restricted to HPA-1b1b and HLA-DRB3*0101-positive pregnant women with an HPA-1a-positive fetus. We investigated whether or not HPA-1a antigen-specific peptides that formed the T cell epitope could reduce IgG anti-HPA-1a responses, using a mouse model we had developed previously. Peripheral blood mononuclear cells (PBMC) in blood donations from HPA-1a-immunized women were injected intraperitoneally (i.p.) into severe combined immunodeficient (SCID) mice with peptides and HPA-1a-positive platelets. Human anti-HPA-1a in murine plasma was quantitated at intervals up to 15 weeks. HPA-1a-specific T cells in PBMC were identified by proliferation assays. Using PBMC of three donors who had little T cell reactivity to HPA-1a peptides in vitro, stimulation of anti-HPA-1a responses by these peptides occurred in vivo. However, with a second donation from one of these women which, uniquely, had high HPA-1a-specific T cell proliferation in vitro, marked suppression of the anti-HPA-1a response by HPA-1a peptides occurred in vivo. HPA-1a peptide immunotherapy in this model depended upon reactivation of HPA-1a T cell responses in the donor. For FNAIT, we suggest that administration of antigen-specific peptides to pregnant women might cause either enhancement or reduction of pathogenic antibodies. PMID:24261689

Low-dose cyclophosphamide administered as daily or single dose enhances the antitumor effects of a therapeutic HPV vaccine

PubMed Central

Peng, Shiwen; Lyford-Pike, Sofia; Akpeng, Belinda; Wu, Annie; Hung, Chien-Fu; Hannaman, Drew; Saunders, John R.; Wu, T.-C.

2012-01-01

Although therapeutic HPV vaccines are able to elicit systemic HPV-specific immunity, clinical responses have not always correlated with levels of vaccine-induced CD8+ T cells in human clinical trials. This observed discrepancy may be attributable to an immunosuppressive tumor microenvironment in which the CD8+ T cells are recruited. Regulatory T cells (Tregs) are cells that can dampen cytotoxic CD8+ T-cell function. Cyclophosphamide (CTX) is a systemic chemotherapeutic agent, which can eradicate immune cells, including inhibitory Tregs. The optimal dose and schedule of CTX administration in combination with immunotherapy to eliminate the Treg population without adversely affecting vaccine-induced T-cell responses is unknown. Therefore, we investigated various dosing and administration schedules of CTX in combination with a therapeutic HPV vaccine in a preclinical tumor model. HPV tumor-bearing mice received either a single preconditioning dose or a daily dose of CTX in combination with the pNGVL4a-CRT/E7(detox) DNA vaccine. Both single and daily dosing of CTX in combination with vaccine had a synergistic anti-tumor effect as compared to monotherapy alone. The potent antitumor responses were attributed to the reduction in Treg frequency and increased infiltration of HPV16 E7-specific CD8+ T cells, which led to higher ratios of CD8+/Treg and CD8+/CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs). There was an observed trend toward decreased vaccine-induced CD8+ T-cell frequency with daily dosing of CTX. We recommend a single, preconditioning dose of CTX prior to vaccination due to its efficacy, ease of administration, and reduced cumulative adverse effect on vaccine-induced T cells. PMID:23011589

Cell cycle perturbation induced by gemcitabine in human tumor cells in cell culture, xenografts and bladder cancer patients: implications for clinical trial designs combining gemcitabine with a Chk1 inhibitor.

PubMed

Montano, Ryan; Khan, Nadeem; Hou, Huagang; Seigne, John; Ernstoff, Marc S; Lewis, Lionel D; Eastman, Alan

2017-09-15

Gemcitabine irreversibly inhibits ribonucleotide reductase and induces S phase arrest but whether this occurs in tumors in mice or patients has not been established. Tumor cells in culture were incubated with gemcitabine for 6 h to approximate the administration schedule in a patient. Concentrations that induced persistent S phase arrest thereafter correlated with cell killing. Administration of gemcitabine to mice also demonstrated a persistent S phase arrest in their tumor. The minimum dose that induced almost complete S phase arrest after 24 h (40 mg/kg) was well below the maximum tolerated dose in mice. S phase arrest was also observed in tumors of bladder cancer patients receiving gemcitabine. The Chk1 inhibitor MK-8776 sensitized cells to gemcitabine with the greatest cell killing when added 18 h after gemcitabine. In mice, the administration of MK-8776 18 h after gemcitabine elicited positivity for the DNA damage marker γH2AX; this also occurred at relatively low dose (40 mg/kg) gemcitabine. Hence, in both cell culture and xenografts, MK-8776 can markedly enhance cell killing of cells reversibly arrested in S phase by gemcitabine. Some cell lines are hypersensitive to MK-8776 as monotherapy, but this was not observed in xenograft models. Effective monotherapy requires a higher dose of Chk1 inhibitor, and target inhibition over a longer time period as compared to its use in combination. These results have important implications for combining Chk1 inhibitors with gemcitabine and suggest that Chk1 inhibitors with increased bioavailability may have improved efficacy both in combination and as monotherapy.

Merkel Cell Polyomavirus Small T Antigen Induces Cancer and Embryonic Merkel Cell Proliferation in a Transgenic Mouse Model.

PubMed

Shuda, Masahiro; Guastafierro, Anna; Geng, Xuehui; Shuda, Yoko; Ostrowski, Stephen M; Lukianov, Stefan; Jenkins, Frank J; Honda, Kord; Maricich, Stephen M; Moore, Patrick S; Chang, Yuan

2015-01-01

Merkel cell polyomavirus (MCV) causes the majority of human Merkel cell carcinomas (MCC) and encodes a small T (sT) antigen that transforms immortalized rodent fibroblasts in vitro. To develop a mouse model for MCV sT-induced carcinogenesis, we generated transgenic mice with a flox-stop-flox MCV sT sequence homologously recombined at the ROSA locus (ROSAsT), allowing Cre-mediated, conditional MCV sT expression. Standard tamoxifen (TMX) administration to adult UbcCreERT2; ROSAsT mice, in which Cre is ubiquitously expressed, resulted in MCV sT expression in multiple organs that was uniformly lethal within 5 days. Conversely, most adult UbcCreERT2; ROSAsT mice survived low-dose tamoxifen administration but developed ear lobe dermal hyperkeratosis and hypergranulosis. Simultaneous MCV sT expression and conditional homozygous p53 deletion generated multi-focal, poorly-differentiated, highly anaplastic tumors in the spleens and livers of mice after 60 days of TMX treatment. Mouse embryonic fibroblasts from these mice induced to express MCV sT exhibited anchorage-independent cell growth. To examine Merkel cell pathology, MCV sT expression was also induced during mid-embryogenesis in Merkel cells of Atoh1CreERT2/+; ROSAsT mice, which lead to significantly increased Merkel cell numbers in touch domes at late embryonic ages that normalized postnatally. Tamoxifen administration to adult Atoh1CreERT2/+; ROSAsT and Atoh1CreERT2/+; ROSAsT; p53flox/flox mice had no effects on Merkel cell numbers and did not induce tumor formation. Taken together, these results show that MCV sT stimulates progenitor Merkel cell proliferation in embryonic mice and is a bona fide viral oncoprotein that induces full cancer cell transformation in the p53-null setting.

The effect of in-vivo intravenous administration of sodium meglumine diatrizoate on some haematological parameters.

PubMed

Agwu, K K; Mgbor, S; Ogbu, S O I; Okeji, M

2007-01-01

To investigate the in-vivo effects of intravenous administration of sodium meglumine diatrizoate on some haematological parameters in a Nigerian population. Blood samples were collected before and one hour after intravenous injection of sodium-meglumine diatrizoate from 50 subjects undergoing intravenous urography examinations who had no history of and laboratory confirmed diseases that may affect haematological parameters. Standard laboratory methods were used to assay the haemoglobin concentration (Hb), packed cell volume (PCV), total white blood cell (WBC) count and differentials and blood film for any morphological changes in the red blood cells (RBC). Comparisons were made between the mean values of these haematological parameters before and one hour post injection using paired t-test for any statistically significant differences. There were statistically significant reductions in the mean values of Hb concentration and the neutrophil count one hour post injection compared with their pretest values (p < 0.05). The lymphocytes were also significantly increased post injection compared to the pretest values whereas 70% of the erythrocytes were morphologically altered from their approximately 100% normocytic shape at pre-test. Intravenous administration of sodium-meglumine diatrizoate causes in-vivo reduction in Hb concentration and neutrophil count in humans as well as poikilocytosis of the erythrocytes. Some of these effects have the potential of triggering or exacerbating crisis in a sickle cell anaemia subject which is endemic in our locality. Caution should therefore be exercised in the choice and administration of radiological contrast agents to sickle cell subjects. Preparations that are iso-osmolar with plasma and have less probability in precipitating crises should be preferred instead.

Antitumor effects of Marginisporum crassissimum (Rhodophyceae), a marine red alga.

PubMed

Hiroishi, S; Sugie, K; Yoshida, T; Morimoto, J; Taniguchi, Y; Imai, S; Kurebayashi, J

2001-06-26

Marginisporum crassissimum (Yendo) Ganesan, a marine red alga found in the ordinal coastal sea around Japan, revealed antitumor (antimetastatic) effects in vitro and in vivo. In in vitro experiments, extracts of this alga inhibited not only the growth of several tumor cell lines, such as B16-BL6 (a mouse melanoma cell line), JYG-B (a mouse mammary carcinoma cell line) and KPL-1 (a human mammary carcinoma cell line), but also invasion of B16-BL6 cells in a culture system. In in vivo experiments, the lung metastasis of B16-BL6 cells inoculated to the tail vein of B57BL/6J mice was inhibited by intraperitoneal administration of an extract from the alga. In addition, life prolongation of B57BL/6J mice inoculated with B16-BL6 cells was also observed by the intraperitoneal administration of the extract. An effective substance showing B16-BL6 growth inhibition in vitro was partially purified by filtration and hydrophobic column chromatography, and was revealed to be sensitive to trypsin-digestion and heat-treatment. The molecular weight of the substance was greater than 100 kDa. This is the first study demonstrating antitumor (antimetastatic) effects of M. crassissimum.

Effect of dark-colored maple syrup on cell proliferation of human gastrointestinal cancer cell

PubMed Central

Yamamoto, Tetsushi; Sato, Kanta; Kubota, Yuika; Mitamura, Kuniko; Taga, Atsushi

2017-01-01

Maple syrup is a natural sweetener that is commonly consumed worldwide. While maple syrup mainly comprises sucrose, it also contains phytochemicals that present various biological effects. Maple syrup is made by boiling down sap, and its color and composition vary in accordance with the sap collection season. Typically, seasonal progression is associated with darker syrup color, and antioxidant activity is proportional to the increasingly dark color. The authors previously reported that maple syrup demonstrated inhibitory effects on colorectal cancer cell growth and invasion, which correlated with darker maple syrup color. In the present study, they examined the effects of two different grades of maple syrup on gastrointestinal cancer cell proliferation, to investigate whether the dark-color maple syrup was suitable as a phytomedicine for gastrointestinal cancer treatment. Administration of dark-color maple syrup significantly inhibited gastrointestinal cancer cell growth as compared to non-treated cancer cells. Moreover, administration of dark-color maple syrup clearly inhibited protein kinase B (AKT) phosphorylation and did not impact mitogen-associated protein kinase phosphorylation. These data suggested that dark-color maple syrup may inhibit cell proliferation through suppression of AKT activation and, thus, may be suitable as a phytomedicine for gastrointestinal cancer treatment. PMID:28685052

Effect of dark-colored maple syrup on cell proliferation of human gastrointestinal cancer cell.

PubMed

Yamamoto, Tetsushi; Sato, Kanta; Kubota, Yuika; Mitamura, Kuniko; Taga, Atsushi

2017-07-01

Maple syrup is a natural sweetener that is commonly consumed worldwide. While maple syrup mainly comprises sucrose, it also contains phytochemicals that present various biological effects. Maple syrup is made by boiling down sap, and its color and composition vary in accordance with the sap collection season. Typically, seasonal progression is associated with darker syrup color, and antioxidant activity is proportional to the increasingly dark color. The authors previously reported that maple syrup demonstrated inhibitory effects on colorectal cancer cell growth and invasion, which correlated with darker maple syrup color. In the present study, they examined the effects of two different grades of maple syrup on gastrointestinal cancer cell proliferation, to investigate whether the dark-color maple syrup was suitable as a phytomedicine for gastrointestinal cancer treatment. Administration of dark-color maple syrup significantly inhibited gastrointestinal cancer cell growth as compared to non-treated cancer cells. Moreover, administration of dark-color maple syrup clearly inhibited protein kinase B (AKT) phosphorylation and did not impact mitogen-associated protein kinase phosphorylation. These data suggested that dark-color maple syrup may inhibit cell proliferation through suppression of AKT activation and, thus, may be suitable as a phytomedicine for gastrointestinal cancer treatment.

Dehydroandrographolide, an iNOS inhibitor, extracted from Andrographis paniculata (Burm.f.) Nees, induces autophagy in human oral cancer cells.

PubMed

Hsieh, Ming-Ju; Lin, Chiao-Wen; Chiou, Hui-Ling; Yang, Shun-Fa; Chen, Mu-Kuan

2015-10-13

Autophagy, which is constitutively executed at the basal level in all cells, promotes cellular homeostasis by regulating the turnover of organelles and proteins. Andrographolide and dehydroandrographolide (DA) are the two principle components of Andrographis paniculata (Burm.f.) Nees. and are the main contributors to its therapeutic properties. However, the pharmacological activities of dehydroandrographolide (DA) remain unclear. In this study, DA induces oral cancer cell death by activating autophagy. Treatment with autophagy inhibitors inhibited DA-induced human oral cancer cell death. In addition, DA increased LC3-II expression and reduced p53 expression in a time- and concentration-dependent manner. Furthermore, DA induced autophagy and decreased cell viability through modulation of p53 expression. DA-induced autophagy was triggered by an activation of JNK1/2 and an inhibition of Akt and p38. In conclusion, this study demonstrated that DA induced autophagy in human oral cancer cells by modulating p53 expression, activating JNK1/2, and inhibiting Akt and p38. Finally, an administration of DA effectively suppressed the tumor formation in the oral carcinoma xenograft model in vivo. This is the first study to reveal the novel function of DA in activating autophagy, suggesting that DA could serve as a new and potential chemopreventive agent for treating human oral cancer.

Dehydroandrographolide, an iNOS inhibitor, extracted from from Andrographis paniculata (Burm.f.) Nees, induces autophagy in human oral cancer cells

PubMed Central

Hsieh, Ming-Ju; Lin, Chiao-Wen; Chiou, Hui-Ling; Yang, Shun-Fa; Chen, Mu-Kuan

2015-01-01

Autophagy, which is constitutively executed at the basal level in all cells, promotes cellular homeostasis by regulating the turnover of organelles and proteins. Andrographolide and dehydroandrographolide (DA) are the two principle components of Andrographis paniculata (Burm.f.) Nees. and are the main contributors to its therapeutic properties. However, the pharmacological activities of dehydroandrographolide (DA) remain unclear. In this study, DA induces oral cancer cell death by activating autophagy. Treatment with autophagy inhibitors inhibited DA-induced human oral cancer cell death. In addition, DA increased LC3-II expression and reduced p53 expression in a time- and concentration-dependent manner. Furthermore, DA induced autophagy and decreased cell viability through modulation of p53 expression. DA-induced autophagy was triggered by an activation of JNK1/2 and an inhibition of Akt and p38. In conclusion, this study demonstrated that DA induced autophagy in human oral cancer cells by modulating p53 expression, activating JNK1/2, and inhibiting Akt and p38. Finally, an administration of DA effectively suppressed the tumor formation in the oral carcinoma xenograft model in vivo. This is the first study to reveal the novel function of DA in activating autophagy, suggesting that DA could serve as a new and potential chemopreventive agent for treating human oral cancer. PMID:26356821

Inhibition of colony-stimulating-factor-1 signaling in vivo with the orally bioavailable cFMS kinase inhibitor GW2580.

PubMed

Conway, James G; McDonald, Brad; Parham, Janet; Keith, Barry; Rusnak, David W; Shaw, Eva; Jansen, Marilyn; Lin, Peiyuan; Payne, Alan; Crosby, Renae M; Johnson, Jennifer H; Frick, Lloyd; Lin, Min-Hwa Jasmine; Depee, Scott; Tadepalli, Sarva; Votta, Bart; James, Ian; Fuller, Karen; Chambers, Timothy J; Kull, Frederick C; Chamberlain, Stanley D; Hutchins, Jeff T

2005-11-01

Colony-stimulating-factor-1 (CSF-1) signaling through cFMS receptor kinase is increased in several diseases. To help investigate the role of cFMS kinase in disease, we identified GW2580, an orally bioavailable inhibitor of cFMS kinase. GW2580 completely inhibited human cFMS kinase in vitro at 0.06 microM and was inactive against 26 other kinases. GW2580 at 1 microM completely inhibited CSF-1-induced growth of mouse M-NFS-60 myeloid cells and human monocytes and completely inhibited bone degradation in cultures of human osteoclasts, rat calvaria, and rat fetal long bone. In contrast, GW2580 did not affect the growth of mouse NS0 lymphoblastoid cells, human endothelial cells, human fibroblasts, or five human tumor cell lines. GW2580 also did not affect lipopolysaccharide (LPS)-induced TNF, IL-6, and prostaglandin E2 production in freshly isolated human monocytes and mouse macrophages. After oral administration, GW2580 blocked the ability of exogenous CSF-1 to increase LPS-induced IL-6 production in mice, inhibited the growth of CSF-1-dependent M-NFS-60 tumor cells in the peritoneal cavity, and diminished the accumulation of macrophages in the peritoneal cavity after thioglycolate injection. Unexpectedly, GW2580 inhibited LPS-induced TNF production in mice, in contrast to effects on monocytes and macrophages in vitro. In conclusion, GW2580's selective inhibition of monocyte growth and bone degradation is consistent with cFMS kinase inhibition. The ability of GW2580 to chronically inhibit CSF-1 signaling through cFMS kinase in normal and tumor cells in vivo makes GW2580 a useful tool in assessing the role of cFMS kinase in normal and disease processes.

Peptide mimic for influenza vaccination using nonnatural combinatorial chemistry

PubMed Central

Miles, John J.; Tan, Mai Ping; Dolton, Garry; Galloway, Sarah A.E.; Laugel, Bruno; Makinde, Julia; Matthews, Katherine K.; Watkins, Thomas S.; Wong, Yide; Clark, Richard J.; Pentier, Johanne M.; Attaf, Meriem; Lissina, Anya; Ager, Ann; Gallimore, Awen; Gras, Stephanie; Rossjohn, Jamie; Burrows, Scott R.; Cole, David K.; Price, David A.

2018-01-01

Polypeptide vaccines effectively activate human T cells but suffer from poor biological stability, which confines both transport logistics and in vivo therapeutic activity. Synthetic biology has the potential to address these limitations through the generation of highly stable antigenic “mimics” using subunits that do not exist in the natural world. We developed a platform based on D–amino acid combinatorial chemistry and used this platform to reverse engineer a fully artificial CD8+ T cell agonist that mirrored the immunogenicity profile of a native epitope blueprint from influenza virus. This nonnatural peptide was highly stable in human serum and gastric acid, reflecting an intrinsic resistance to physical and enzymatic degradation. In vitro, the synthetic agonist stimulated and expanded an archetypal repertoire of polyfunctional human influenza virus–specific CD8+ T cells. In vivo, specific responses were elicited in naive humanized mice by subcutaneous vaccination, conferring protection from subsequent lethal influenza challenge. Moreover, the synthetic agonist was immunogenic after oral administration. This proof-of-concept study highlights the power of synthetic biology to expand the horizons of vaccine design and therapeutic delivery. PMID:29528337

Human Gut-Derived Commensal Bacteria Suppress CNS Inflammatory and Demyelinating Disease.

PubMed

Mangalam, Ashutosh; Shahi, Shailesh K; Luckey, David; Karau, Melissa; Marietta, Eric; Luo, Ningling; Choung, Rok Seon; Ju, Josephine; Sompallae, Ramakrishna; Gibson-Corley, Katherine; Patel, Robin; Rodriguez, Moses; David, Chella; Taneja, Veena; Murray, Joseph

2017-08-08

The human gut is colonized by a large number of microorganisms (∼10 13 bacteria) that support various physiologic functions. A perturbation in the healthy gut microbiome might lead to the development of inflammatory diseases, such as multiple sclerosis (MS). Therefore, gut commensals might provide promising therapeutic options for treating MS and other diseases. We report the identification of human gut-derived commensal bacteria, Prevotella histicola, which can suppress experimental autoimmune encephalomyelitis (EAE) in a human leukocyte antigen (HLA) class II transgenic mouse model. P. histicola suppresses disease through the modulation of systemic immune responses. P. histicola challenge led to a decrease in pro-inflammatory Th1 and Th17 cells and an increase in the frequencies of CD4 + FoxP3 + regulatory T cells, tolerogenic dendritic cells, and suppressive macrophages. Our study provides evidence that the administration of gut commensals may regulate a systemic immune response and may, therefore, have a possible role in treatment strategies for MS. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Cytoskeletal defects in Bmpr2-associated pulmonary arterial hypertension.

PubMed

Johnson, Jennifer A; Hemnes, Anna R; Perrien, Daniel S; Schuster, Manfred; Robinson, Linda J; Gladson, Santhi; Loibner, Hans; Bai, Susan; Blackwell, Tom R; Tada, Yuji; Harral, Julie W; Talati, Megha; Lane, Kirk B; Fagan, Karen A; West, James

2012-03-01

The heritable form of pulmonary arterial hypertension (PAH) is typically caused by a mutation in bone morphogenic protein receptor type 2 (BMPR2), and mice expressing Bmpr2 mutations develop PAH with features similar to human disease. BMPR2 is known to interact with the cytoskeleton, and human array studies in PAH patients confirm alterations in cytoskeletal pathways. The goal of this study was to evaluate cytoskeletal defects in BMPR2-associated PAH. Expression arrays on our Bmpr2 mutant mouse lungs revealed cytoskeletal defects as a prominent molecular consequence of universal expression of a Bmpr2 mutation (Rosa26-Bmpr2(R899X)). Pulmonary microvascular endothelial cells cultured from these mice have histological and functional cytoskeletal defects. Stable transfection of different BMPR2 mutations into pulmonary microvascular endothelial cells revealed that cytoskeletal defects are common to multiple BMPR2 mutations and are associated with activation of the Rho GTPase, Rac1. Rac1 defects are corrected in cell culture and in vivo through administration of exogenous recombinant human angiotensin-converting enzyme 2 (rhACE2). rhACE2 reverses 77% of gene expression changes in Rosa26-Bmpr2(R899X) transgenic mice, in particular, correcting defects in cytoskeletal function. Administration of rhACE2 to Rosa26-Bmpr2(R899X) mice with established PAH normalizes pulmonary pressures. Together, these findings suggest that cytoskeletal function is central to the development of BMPR2-associated PAH and that intervention against cytoskeletal defects may reverse established disease.

Granzyme M and K release in human experimental endotoxemia.

PubMed

Wensink, Annette C; Wiewel, Maryse A; Jongeneel, Lieneke H; Boes, Marianne; van der Poll, Tom; Hack, C Erik; Bovenschen, Niels

2016-07-01

Granzymes are serine proteases involved in killing of tumor cells and virally infected cells. However, granzymes are also upregulated in blood under inflammatory conditions and contribute to cytokine release and processing. Here, we show that granzyme M (GrM) and to a lesser extent GrK are transiently elevated in the circulation following LPS administration in humans. GrM is released upon stimulation of whole blood with LPS or the gram-negative bacteria Escherichia coli BL21, Pseudomonas aeruginosa, and Neisseria meningitidis. GrK is only released upon stimulation with P. aeruginosa. Thus, GrM and GrK are differentially released in response to LPS and gram-negative bacteria. Copyright © 2016 Elsevier GmbH. All rights reserved.

Development of a new live attenuated mumps virus vaccine in human diploid cells.

PubMed

Sassani, A; Mirchamsy, H; Shafyi, A; Ahourai, P; Razavi, J; Gholami, M R; Mohammadi, A; Ezzi, A; Rahmani, M; Fateh, G

1991-07-01

A new live attenuated mumps vaccine was developed in human diploid cells. The S-12 virus was isolated from a 10-year-old girl showing typical symptoms of mumps infection, the diagnosis was confirmed by a pediatrician. The virus was isolated in green monkey kidney cells, without passage in chick embryo cavity or chick embryo fibroblasts. Attenuation of the wild virus was performed by serial passages in human diploid cells (MRC-5). The attenuated virus was characterized by identity tests, as well as by a reduction in plaque size, as marker tests. The virus was free from adventitious agents and safe for laboratory animals as well as for monkeys. The reactogenicity and immunogenicity of the S-12 virus for man was investigated by administration of a monovalent vaccine to 20 seronegative adult male volunteers and 30 children aged 1 to 5 years without history of mumps infection or vaccination. Seroconversion was obtained in 95% of the vaccinees. The new vaccine has the advantage of not requiring specific pathogen-free eggs, and being free from avian proteins and therefore can be used in sensitized patients.

AAV capsid CD8+ T-cell epitopes are highly conserved across AAV serotypes

PubMed Central

Hui, Daniel J; Edmonson, Shyrie C; Podsakoff, Gregory M; Pien, Gary C; Ivanciu, Lacramioara; Camire, Rodney M; Ertl, Hildegund; Mingozzi, Federico; High, Katherine A; Basner-Tschakarjan, Etiena

2015-01-01

Adeno-associated virus (AAV) has become one of the most promising vectors in gene transfer in the last 10 years with successful translation to clinical trials in humans and even market approval for a first gene therapy product in Europe. Administration to humans, however, revealed that adaptive immune responses against the vector capsid can present an obstacle to sustained transgene expression due to the activation and expansion of capsid-specific T cells. The limited number of peripheral blood mononuclear cells (PBMCs) obtained from samples within clinical trials allows for little more than monitoring of T-cell responses. We were able to identify immunodominant major histocompatibility complex (MHC) class I epitopes for common human leukocyte antigen (HLA) types by using spleens isolated from subjects undergoing splenectomy for non-malignant indications as a source of large numbers of lymphocytes and restimulating them with single AAV capsid peptides in vitro. Further experiments confirmed that these epitopes are naturally processed and functionally relevant. The design of more effective and less immunogenic AAV vectors, and precise immune monitoring of vector-infused subjects, are facilitated by these findings. PMID:26445723

AAV capsid CD8+ T-cell epitopes are highly conserved across AAV serotypes.

PubMed

Hui, Daniel J; Edmonson, Shyrie C; Podsakoff, Gregory M; Pien, Gary C; Ivanciu, Lacramioara; Camire, Rodney M; Ertl, Hildegund; Mingozzi, Federico; High, Katherine A; Basner-Tschakarjan, Etiena

2015-01-01

Adeno-associated virus (AAV) has become one of the most promising vectors in gene transfer in the last 10 years with successful translation to clinical trials in humans and even market approval for a first gene therapy product in Europe. Administration to humans, however, revealed that adaptive immune responses against the vector capsid can present an obstacle to sustained transgene expression due to the activation and expansion of capsid-specific T cells. The limited number of peripheral blood mononuclear cells (PBMCs) obtained from samples within clinical trials allows for little more than monitoring of T-cell responses. We were able to identify immunodominant major histocompatibility complex (MHC) class I epitopes for common human leukocyte antigen (HLA) types by using spleens isolated from subjects undergoing splenectomy for non-malignant indications as a source of large numbers of lymphocytes and restimulating them with single AAV capsid peptides in vitro. Further experiments confirmed that these epitopes are naturally processed and functionally relevant. The design of more effective and less immunogenic AAV vectors, and precise immune monitoring of vector-infused subjects, are facilitated by these findings.

N-acetylcysteine-pretreated human embryonic mesenchymal stem cell administration protects against bleomycin-induced lung injury.

PubMed

Wang, Qiao; Zhu, Hong; Zhou, Wu-Gang; Guo, Xiao-Can; Wu, Min-Juan; Xu, Zhen-Yu; Jiang, Jun-feng; Shen, Ce; Liu, Hou-Qi

2013-08-01

The transplantation of mesenchymal stem cells (MSCs) has been reported to be a promising approach in the treatment of acute lung injury. However, the poor efficacy of transplanted MSCs is one of the serious handicaps in the progress of MSC-based therapy. Therefore, the purpose of this study was to investigate whether the pretreatment of human embryonic MSCs (hMSCs) with an antioxidant, namely N-acetylcysteine (NAC), can improve the efficacy of hMSC transplantation in lung injury. In vitro, the antioxidant capacity of NAC-pretreated hMSCs was assessed using intracellular reactive oxygen species (ROS) and glutathione assays and cell adhesion and spreading assays. In vivo, the therapeutic potential of NAC-pretreated hMSCs was assessed in a bleomycin-induced model of lung injury in nude mice. The pretreatment of hMSCs with NAC improved antioxidant capacity to defend against redox imbalances through the elimination of cellular ROS, increasing cellular glutathione levels, and the enhancement of cell adhesion and spreading when exposed to oxidative stresses in vitro. In addition, the administration of NAC-pretreated hMSCs to nude mice with bleomycin-induced lung injury decreased the pathological grade of lung inflammation and fibrosis, hydroxyproline content and numbers of neutrophils and inflammatory cytokines in bronchoalveolar lavage fluid and apoptotic cells, while enhancing the retention and proliferation of hMSCs in injured lung tissue and improving the survival rate of mice compared with results from untreated hMSCs. The pretreatment of hMSCs with NAC could be a promising therapeutic approach to improving cell transplantation and, therefore, the treatment of lung injury.

Human Dental Pulp Stem Cells Are More Effective Than Human Bone Marrow-Derived Mesenchymal Stem Cells in Cerebral Ischemic Injury.

PubMed

Song, Miyeoun; Lee, Jae-Hyung; Bae, Jinhyun; Bu, Youngmin; Kim, Eun-Cheol

2017-06-09

We compared the therapeutic effects and mechanism of transplanted human dental pulp stem cells (hDPSCs) and human bone marrow-derived mesenchymal stem cells (hBM-MSCs) in a rat stroke model and an in vitro model of ischemia. Rats were intravenously injected with hDPSCs or hBM-MSCs 24 h after middle cerebral artery occlusion (MCAo), and both groups showed improved functional recovery and reduced infarct volume versus control rats, but the hDPSC group showed greater reduction in infarct volume than the hBM-MSC group. The positive area for the endothelial cell marker was greater in the lesion boundary areas in the hDPSC group than in the hBM-MSC group. Administration of hDPSCs to rats with stroke significantly decreased reactive gliosis, as evidenced by the attenuation of MCAo-induced GFAP+/nestin+ and GFAP+/Musashi-1+ cells, compared with hBM-MSCs. In vivo findings were confirmed by in vitro data illustrating that hDPSCs showed superior neuroprotective, migratory, and in vitro angiogenic effects in oxygen-glucose deprivation (OGD)-injured human astrocytes (hAs) versus hBM-MSCs. Comprehensive comparative bioinformatics analyses from hDPSC- and hBM-MSC-treated in vitro OGD-injured hAs were examined by RNA sequencing technology. In gene ontology and KEGG pathway analyses, significant pathways in the hDPSC-treated group were the MAPK and TGF-β signaling pathways. Thus, hDPSCs may be a better cell therapy source for ischemic stroke than hBM-MSCs.

Noninvasive Imaging of Administered Progenitor Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steven R Bergmann, M.D., Ph.D.

The objective of this research grant was to develop an approach for labeling progenitor cells, specifically those that we had identified as being able to replace ischemic heart cells, so that the distribution could be followed non-invasively. In addition, the research was aimed at determining whether administration of progenitor cells resulted in improved myocardial perfusion and function. The efficiency and toxicity of radiolabeling of progenitor cells was to be evaluated. For the proposed clinical protocol, subjects with end-stage ischemic coronary artery disease were to undergo a screening cardiac positron emission tomography (PET) scan using N-13 ammonia to delineate myocardial perfusionmore » and function. If they qualified based on their PET scan, they would undergo an in-hospital protocol whereby CD34+ cells were stimulated by the administration of granulocytes-colony stimulating factor (G-CSF). CD34+ cells would then be isolated by apharesis, and labeled with indium-111 oxine. Cells were to be re-infused and subjects were to undergo single photon emission computed tomography (SPECT) scanning to evaluate uptake and distribution of labeled progenitor cells. Three months after administration of progenitor cells, a cardiac PET scan was to be repeated to evaluate changes in myocardial perfusion and/or function. Indium oxine is a radiopharmaceutical for labeling of autologous lymphocytes. Indium-111 (In-111) decays by electron capture with a t{sub ½} of 67.2 hours (2.8 days). Indium forms a saturated complex that is neutral, lipid soluble, and permeates the cell membrane. Within the cell, the indium-oxyquinolone complex labels via indium intracellular chelation. Following leukocyte labeling, ~77% of the In-111 is incorporated in the cell pellet. The presence of red cells and /or plasma reduces the labeling efficacy. Therefore, the product needed to be washed to eliminate plasma proteins. This repeated washing can damage cells. The CD34 selected product was a 90-99% pure population of leukocytes. Viability was assessed using Trypan blue histological analysis. We successfully isolated and labeled ~25-30 x 10{sup 7} CD34+ lymphocytes in cytokine mobilized progenitor cell apharesis harvests. Cells were also subjected to a stat gram stain to look for bacterial contamination, stat endotoxin LAL to look for endotoxin contamination, flow cytometry for evaluation of the purity of the cells and 14-day sterility culture. Colony forming assays confirm the capacity of these cells to proliferate and function ex-vivo with CFU-GM values of 26 colonies/ 1 x 10{sup 4} cells plated and 97% viability in cytokine augmented methylcellulose at 10-14 days in CO{sub 2} incubation. We developed a closed-processing system for the product labeling prior to infusion to maintain autologous cell integrity and sterility. Release criteria for the labeled product were documented for viability, cell count and differential, and measured radiolabel. We were successful in labeling the cells with up to 500 uCi/10{sup 8} cells, with viability of >98%. However, due to delays in getting the protocol approved by the FDA, the cells were not infused in humans in this location (although we did successfully use CD34+ cells in humans in a study in Australia). The approach developed should permit labeling of progenitor cells that can be administered to human subjects for tracking. The labeling approach should be useful for all progenitor cell types, although this would need to be verified since different cell lines may have differential radiosensitivity.« less

Identification of DNA-PKcs as a primary resistance factor of TIC10 in hepatocellular carcinoma cells.

PubMed

Cheng, Long; Liu, Yuan-Yuan; Lu, Pei-Hua; Peng, Yi; Yuan, Qiang; Gu, Xin-Shi; Jin, Yong; Chen, Min-Bin; Bai, Xu-Ming

2017-04-25

The current study tested the anti-hepatocellular carcinoma (HCC) cell activity of TIC10, a first-in-class small-molecule tumor necrosis (TNF)-related apoptosis-inducing ligand (TRAIL) inducer. TIC10 exerted potent anti-proliferative and pro-apoptotic actions in primary and established human HCC cells. TIC10 blocked Akt-Erk activation, leading to Foxo3a nuclear translocation, as well as TRAIL and death receptor-5 (DR5) transcription in HCC cells. We propose that DNA-PKcs is a major resistance factor of TIC10 possibly via inhibiting Foxo3a nuclear translocation. DNA-PKcs inhibition, knockdown or mutation facilitated TIC10-induced Foxo3a nuclear translocation, TRAIL/DR5 expression and cell apoptosis. Reversely, exogenous DNA-PKcs over-expression inhibited above actions by TIC10. In vivo, oral administration of TIC10 significantly inhibited HepG2 tumor growth in nude mice, which was further potentiated with Nu7026 co-administration. Thus, TIC10 shows promising anti-HCC activity, alone or together with DNA-PKcs inhibitors.

Recombinant human SDF-1α administration accelerates aneurysm neck reendothelialization in rabbit saccular aneurysm after flow diverter treatment.

PubMed

Li, Zifu; Zhao, Rui; Fang, Xinggen; Huang, Qinghai; Liu, Jianmin

2017-03-01

Reendothelialization in the aneurysm neck is pivotal to vascular repair for intracranial aneurysm after flow diverter (FD) implantation. Recombinant human stromal cell-derived factor 1α (rhSDF-1α) is a vital chemoattractant to stem cells and potentially facilitates reendothelialization. Here, we sought to investigate the therapeutic effects of intravenous administration of rhSDF-1α and uncover its potential mechanism for promoting aneurysm neck reendothelialization. Recombinant pET32a-186 plasmid was transformed into Escherichia coli to produce the rhSDF-1α protein with biological activity. FD was implanted into the elastase-induced saccular aneurysm in New Zealand white rabbits. rhSDF-1α (50 μg/kg/day) was intravenously administrated for consecutive 7 days after FD implantation. After these procedures, aneurysms were harvested after 2 or 4 weeks. Scanning electron microscopy was used to measure the neointima thickness and count the endothelial-like cells at aneurysm neck. Four weeks later, the mRNA levels of endothelial markers in the neointima at aneurysm neck were examined. Migration assay showed that rhSDF-1α could induce migration of endothelial progenitor cells in a dose-dependent manner. Two weeks after stent implantation, follow-up angiography showed partial aneurysm occlusion in one of each group and total aneurysm occlusion in 17 saccular aneurysm rabbits (9 of the rhSDF-1α group and 8 of the control group). No significant change of neointima thickness at aneurysm neck was observed. Intriguingly, more endothelial-like cells were observed at aneurysm neck in the rhSDF-1α group at 2 weeks (55 vs 13 cells per high-power field) and 4 weeks (104 vs 60 cells per high-power field). The mRNA levels of Tie-2, VE-cadherin, KDR and E-selectin were significantly enhanced compared with those of the control group. These results showed that intravenous administration of rhSDF-1α can accelerate reendothelialization in the aneurysm neck after FD implantation. Our study reveals an important role of rhSDF-1α in inducing aneurysm occlusion and suggests that it achieves its function through modulating the reendothelialization. © The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Potential transfer of neurotoxic amino acid β-N-methylamino-alanine (BMAA) from mother to infant during breast-feeding: Predictions from human cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Andersson, Marie

β-N-methylamino-alanine (BMAA) is a non-protein amino acid produced by cyanobacteria, diatoms and dinoflagellates. BMAA has potential to biomagnify in a terrestrial food chain, and to bioaccumulate in fish and shellfish. We have reported that administration of [{sup 14}C]L-BMAA to lactating mice and rats results in a mother to off-spring transfer via the milk. A preferential enantiomer-specific uptake of [{sup 14}C]L-BMAA has also been demonstrated in differentiated murine mammary epithelium HC11 cells. These findings, together with neurotoxic effects of BMAA demonstrated both in vitro and in vivo, highlight the need to determine whether such transfer could also occur in humans. Here,more » we used four cell lines of human origin to examine and compare the transport of the two BMAA enantiomers in vitro. The uptake patterns of [{sup 14}C]L- and [{sup 14}C]D-BMAA in the human mammary MCF7 cell line were in agreement with the results in murine HC11 cells, suggesting a potential secretion of BMAA into human breast milk. The permeability coefficients for both [{sup 14}C]L- and [{sup 14}C]D-BMAA over monolayers of human intestinal Caco2 cells supported an efficient absorption from the human intestine. As a final step, transport experiments confirmed that [{sup 14}C]L-and [{sup 14}C]D-BMAA can be taken up by human SHSY5Y neuroblastoma cells and even more efficiently by human U343 glioblastoma cells. In competition experiments with various amino acids, the ASCT2 specific inhibitor benzylserine was the most effective inhibitor of [{sup 14}C]L-BMAA uptake tested here. Altogether, our results suggest that BMAA can be transferred from an exposed mother, via the milk, to the brain of the nursed infant. - Highlights: • Transport of BMAA in human intestinal, mammary and CNS cell lines was examined. • The transport of L-BMAA over intestinal cell monolayers was unidirectional. • Enantiomer-selective uptake of L-BMAA in breast, neuron and glia cells was evident. • Competition experiments indicate that L-BMAA uptake involved several transporters. • A potential for mother to infant transfer of BMAA is proposed.« less

In vitro and in vivo antiproliferative activity of metformin on stem-like cells isolated from spontaneous canine mammary carcinomas: translational implications for human tumors.

PubMed

Barbieri, Federica; Thellung, Stefano; Ratto, Alessandra; Carra, Elisa; Marini, Valeria; Fucile, Carmen; Bajetto, Adriana; Pattarozzi, Alessandra; Würth, Roberto; Gatti, Monica; Campanella, Chiara; Vito, Guendalina; Mattioli, Francesca; Pagano, Aldo; Daga, Antonio; Ferrari, Angelo; Florio, Tullio

2015-04-07

Cancer stem cells (CSCs) are considered the cell subpopulation responsible for breast cancer (BC) initiation, growth, and relapse. CSCs are identified as self-renewing and tumor-initiating cells, conferring resistance to chemo- and radio-therapy to several neoplasias. Nowadays, th (about 10mM)e pharmacological targeting of CSCs is considered an ineludible therapeutic goal. The antidiabetic drug metformin was reported to suppress in vitro and in vivo CSC survival in different tumors and, in particular, in BC preclinical models. However, few studies are available on primary CSC cultures derived from human postsurgical BC samples, likely because of the limited amount of tissue available after surgery. In this context, comparative oncology is acquiring a relevant role in cancer research, allowing the analysis of larger samples from spontaneous pet tumors that represent optimal models for human cancer. Isolation of primary canine mammary carcinoma (CMC) cells and enrichment in stem-like cell was carried out from fresh tumor specimens by culturing cells in stem-permissive conditions. Phenotypic and functional characterization of CMC-derived stem cells was performed in vitro, by assessment of self-renewal, long-lasting proliferation, marker expression, and drug sensitivity, and in vivo, by tumorigenicity experiments. Corresponding cultures of differentiated CMC cells were used as internal reference. Metformin efficacy on CMC stem cell viability was analyzed both in vitro and in vivo. We identified a subpopulation of CMC cells showing human breast CSC features, including expression of specific markers (i.e. CD44, CXCR4), growth as mammospheres, and tumor-initiation in mice. These cells show resistance to doxorubicin but were highly sensitive to metformin in vitro. Finally, in vivo metformin administration significantly impaired CMC growth in NOD-SCID mice, associated with a significant depletion of CSCs. Similarly to the human counterpart, CMCs contain stem-like subpopulations representing, in a comparative oncology context, a valuable translational model for human BC, and, in particular, to predict the efficacy of antitumor drugs. Moreover, metformin represents a potential CSC-selective drug for BC, as effective (neo-)adjuvant therapy to eradicate CSC in mammary carcinomas of humans and animals.

Concordance of preclinical and clinical pharmacology and toxicology of therapeutic monoclonal antibodies and fusion proteins: cell surface targets

PubMed Central

Bugelski, Peter J; Martin, Pauline L

2012-01-01

Monoclonal antibodies (mAbs) and fusion proteins directed towards cell surface targets make an important contribution to the treatment of disease. The purpose of this review was to correlate the clinical and preclinical data on the 15 currently approved mAbs and fusion proteins targeted to the cell surface. The principal sources used to gather data were: the peer reviewed Literature; European Medicines Agency ‘Scientific Discussions’; and the US Food and Drug Administration ‘Pharmacology/Toxicology Reviews’ and package inserts (United States Prescribing Information). Data on the 15 approved biopharmaceuticals were included: abatacept; abciximab; alefacept; alemtuzumab; basiliximab; cetuximab; daclizumab; efalizumab; ipilimumab; muromonab; natalizumab; panitumumab; rituximab; tocilizumab; and trastuzumab. For statistical analysis of concordance, data from these 15 were combined with data on the approved mAbs and fusion proteins directed towards soluble targets. Good concordance with human pharmacodynamics was found for mice receiving surrogates or non-human primates (NHPs) receiving the human pharmaceutical. In contrast, there was poor concordance for human pharmacodynamics in genetically deficient mice and for human adverse effects in all three test systems. No evidence that NHPs have superior predictive value was found. PMID:22168282

Prevention of carcinoma of cervix with human papillomavirus vaccine.

PubMed

Gavarasana, S; Kalasapudi, R S; Rao, T D; Thirumala, S

2000-01-01

Carcinoma of cervix is the most common cancer found among the women of India. Though cervical cytology screening was effective in preventing carcinoma of cervix in developed nations, it is considered unsuitable in developing countries. Recent research has established an etiological link between human papillomavirus infection and carcinoma of cervix. In this review, an attempt is made to answer the question, 'whether carcinoma of cervix can be prevented with human papillomavirus vaccine?' Literature search using Pubmed and Medline was carried out and relevant articles were reviewed. There is ample experimental evidence to show that DNA of human papillomavirus integrates with cervical cell genome. Viral genes E6 and E7 of HPV type 16 and 18 inactivate p53 function and Rb gene, thus immortalize the cervical epithelial cells. Recombinant vaccines blocked the function of E6 and E7 genes preventing development of papillomas in animals. Vaccination with HPV-VLPs encoding for genes of E6 and E7 neutralizes HPV integrated genome of malignant cells of uterine cervix. Based on experimental evidence, it is possible to prevent carcinoma of cervix with human papillomavirus vaccine, Further research is necessary to identify a effective and safe HPV vaccine, routes of administration and characteristics of potential beneficiaries.

Three-dimensional tissue assemblies: novel models for the study of Salmonella enterica serovar Typhimurium pathogenesis

NASA Technical Reports Server (NTRS)

Nickerson, C. A.; Goodwin, T. J.; Terlonge, J.; Ott, C. M.; Buchanan, K. L.; Uicker, W. C.; Emami, K.; LeBlanc, C. L.; Ramamurthy, R.; Clarke, M. S.;

Three-Dimensional Tissue Assemblies: Novel Models for the Study of Salmonella enterica Serovar Typhimurium Pathogenesis

PubMed Central

Nickerson, Cheryl A.; Goodwin, Thomas J.; Terlonge, Jacqueline; Ott, C. Mark; Buchanan, Kent L.; Uicker, William C.; Emami, Kamal; LeBlanc, Carly L.; Ramamurthy, Rajee; Clarke, Mark S.; Vanderburg, Charles R.; Hammond, Timothy; Pierson, Duane L.

2001-01-01

The lack of readily available experimental systems has limited knowledge pertaining to the development of Salmonella-induced gastroenteritis and diarrheal disease in humans. We used a novel low-shear stress cell culture system developed at the National Aeronautics and Space Administration in conjunction with cultivation of three-dimensional (3-D) aggregates of human intestinal tissue to study the infectivity of Salmonella enterica serovar Typhimurium for human intestinal epithelium. Immunohistochemical characterization and microscopic analysis of 3-D aggregates of the human intestinal epithelial cell line Int-407 revealed that the 3-D cells more accurately modeled human in vivo differentiated tissues than did conventional monolayer cultures of the same cells. Results from infectivity studies showed that Salmonella established infection of the 3-D cells in a much different manner than that observed for monolayers. Following the same time course of infection with Salmonella, 3-D Int-407 cells displayed minimal loss of structural integrity compared to that of Int-407 monolayers. Furthermore, Salmonella exhibited significantly lower abilities to adhere to, invade, and induce apoptosis of 3-D Int-407 cells than it did for infected Int-407 monolayers. Analysis of cytokine expression profiles of 3-D Int-407 cells and monolayers following infection with Salmonella revealed significant differences in expression of interleukin 1α (IL-1α), IL-1β, IL-6, IL-1Ra, and tumor necrosis factor alpha mRNAs between the two cultures. In addition, uninfected 3-D Int-407 cells constitutively expressed higher levels of transforming growth factor β1 mRNA and prostaglandin E2 than did uninfected Int-407 monolayers. By more accurately modeling many aspects of human in vivo tissues, the 3-D intestinal cell model generated in this study offers a novel approach for studying microbial infectivity from the perspective of the host-pathogen interaction. PMID:11598087

PEGylated PLGA-based nanoparticles targeting M cells for oral vaccination.

PubMed

Garinot, Marie; Fiévez, Virginie; Pourcelle, Vincent; Stoffelbach, François; des Rieux, Anne; Plapied, Laurence; Theate, Ivan; Freichels, Hélène; Jérôme, Christine; Marchand-Brynaert, Jacqueline; Schneider, Yves-Jacques; Préat, Véronique

2007-07-31

To improve the efficiency of orally delivered vaccines, PEGylated PLGA-based nanoparticles displaying RGD molecules at their surface were designed to target human M cells. RGD grafting was performed by an original method called "photografting" which covalently linked RGD peptides mainly on the PEG moiety of the PCL-PEG, included in the formulation. First, three non-targeted formulations with size and zeta potential adapted to M cell uptake and stable in gastro-intestinal fluids, were developed. Their transport by an in vitro model of the human Follicle associated epithelium (co-cultures) was largely increased as compared to mono-cultures (Caco-2 cells). RGD-labelling of nanoparticles significantly increased their transport by co-cultures, due to interactions between the RGD ligand and the beta(1) intregrins detected at the apical surface of co-cultures. In vivo studies demonstrated that RGD-labelled nanoparticles particularly concentrated in M cells. Finally, ovalbumin-loaded nanoparticles were orally administrated to mice and induced an IgG response, attesting antigen ability to elicit an immune response after oral delivery.

Modified Vaccinia Virus Ankara Preferentially Targets Antigen Presenting Cells In Vitro, Ex Vivo and In Vivo.

PubMed

Altenburg, Arwen F; van de Sandt, Carolien E; Li, Bobby W S; MacLoughlin, Ronan J; Fouchier, Ron A M; van Amerongen, Geert; Volz, Asisa; Hendriks, Rudi W; de Swart, Rik L; Sutter, Gerd; Rimmelzwaan, Guus F; de Vries, Rory D

2017-08-17

Modified Vaccinia virus Ankara (MVA) is a promising vaccine vector with an excellent safety profile. However, despite extensive pre-clinical and clinical testing, surprisingly little is known about the cellular tropism of MVA, especially in relevant animal species. Here, we performed in vitro, ex vivo and in vivo experiments with recombinant MVA expressing green fluorescent protein (rMVA-GFP). In both human peripheral blood mononuclear cells and mouse lung explants, rMVA-GFP predominantly infected antigen presenting cells. Subsequent in vivo experiments performed in mice, ferrets and non-human primates indicated that preferential targeting of dendritic cells and alveolar macrophages was observed after respiratory administration, although subtle differences were observed between the respective animal species. Following intramuscular injection, rMVA-GFP was detected in interdigitating cells between myocytes, but also in myocytes themselves. These data are important in advancing our understanding of the basis for the immunogenicity of MVA-based vaccines and aid rational vaccine design and delivery strategies.

Toxicity and Pharmacokinetic Profile for Single-Dose Injection of ABY-029: a Fluorescent Anti-EGFR Synthetic Affibody Molecule for Human Use.

PubMed

Samkoe, Kimberley S; Gunn, Jason R; Marra, Kayla; Hull, Sally M; Moodie, Karen L; Feldwisch, Joachim; Strong, Theresa V; Draney, Daniel R; Hoopes, P Jack; Roberts, David W; Paulsen, Keith; Pogue, Brian W

2017-08-01

ABY-029, a synthetic Affibody peptide, Z03115-Cys, labeled with a near-infrared fluorophore, IRDye® 800CW, targeting epidermal growth factor receptor (EGFR) has been produced under good manufacturing practices for a US Food and Drug Administration-approved first-in-use human study during surgical resection of glioma, as well as other tumors. Here, the pharmacology, phototoxicity, receptor activity, and biodistribution studies of ABY-029 were completed in rats, prior to the intended human use. Male and female Sprague Dawley rats were administered a single intravenous dose of varying concentrations (0, 245, 2449, and 24,490 μg/kg corresponding to 10×, 100×, and 1000× an equivalent human microdose level) of ABY-029 and observed for up to 14 days. Histopathological assessment of organs and tissues, clinical chemistry, and hematology were performed. In addition, pharmacokinetic clearance and biodistribution of ABY-029 were studied in subgroups of the animals. Phototoxicity and ABY-029 binding to human and rat EGFR were assessed in cell culture and on immobilized receptors, respectively. Histopathological assessment and hematological and clinical chemistry analysis demonstrated that single-dose ABY-029 produced no pathological evidence of toxicity at any dose level. No phototoxicity was observed in EGFR-positive and EGFR-negative glioma cell lines. Binding strength and pharmacokinetics of the anti-EGFR Affibody molecules were retained after labeling with the dye. Based on the successful safety profile of ABY-029, the 1000× human microdose 24.5 mg/kg was identified as the no observed adverse effect level following intravenous administration. Conserved binding strength and no observed light toxicity also demonstrated ABY-029 safety for human use.

Extracellular superoxide dismutase increased the therapeutic potential of human mesenchymal stromal cells in radiation pulmonary fibrosis.

PubMed

Wei, Li; Zhang, Jing; Yang, Zai-Liang; You, Hua

2017-05-01

Pulmonary fibrosis induced by irradiation is a significant problem of radiotherapy in cancer patients. Extracellular superoxide dismutase (SOD3) is found to be predominantly and highly expressed in the extracellular matrix of lung and plays a pivotal role against oxidative damage. Early administration of mesenchymal stromal cells (MSCs) has been demonstrated to reduce fibrosis of damaged lung. However, injection of MSCs at a later stage would be involved in fibrosis development. The present study aimed to determine whether injection of human umbilical cord-derived MSCs (UC-MSCs) over-expressing SOD3 at the established fibrosis stage would have beneficial effects in a mice model of radiation pulmonary fibrosis. Herein, pulmonary fibrosis in mice was induced using Cobalt-60 ( 60 Co) irradiator with 20 Gy, followed by intravenous injection of UC-MSCs, transduced or not to express SOD3 at 2 h (early delivery) and 60 day (late delivery) post-irradiation, respectively. Our results demonstrated that the early administration of UC-MSCs could attenuate the microscopic damage, reduce collagen deposition, inhibit (myo)fibroblast proliferation, reduce inflammatory cell infiltration, protect alveolar type II (AE2) cell injury, prevent oxidative stress and increase antioxidant status, and reduce pro-fibrotic cytokine level in serum. Furthermore, the early treatment with SOD3-infected UC-MSCs resulted in better improvement. However, we failed to observe the therapeutic effects of UC-MSCs, transduced to express SOD3, during established fibrosis. Altogether, our results demonstrated that the early treatment with UC-MSCs alone significantly reduced radiation pulmonary fibrosis in mice through paracrine effects, with further improvement by administration of SOD3-infected UC-MSCs, suggesting that SOD3-infected UC-MSCs may be a potential cell-based gene therapy to treat clinical radiation pulmonary fibrosis. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Defibrotide in combination with granulocyte colony-stimulating factor significantly enhances the mobilization of primitive and committed peripheral blood progenitor cells in mice.

PubMed

Carlo-Stella, Carmelo; Di Nicola, Massimo; Magni, Michele; Longoni, Paolo; Milanesi, Marco; Stucchi, Claudio; Cleris, Loredana; Formelli, Franca; Gianni, Massimo A

2002-11-01

Defibrotide is a polydeoxyribonucleotide, which significantly reduces the expression of adhesion molecules on endothelial cells. We investigated the activity of Defibrotide alone or in combination with recombinant human granulocyte colony-stimulating factor (rhG-CSF) to mobilize peripheral blood progenitor cells (PBPCs) in BALB/c mice. A 5-day treatment with Defibrotide alone (1-15 mg/mouse/day) had no effect on WBC counts, frequencies and absolute numbers of total circulating colony-forming cells (CFCs), i.e., granulocyte-macrophage colony-forming units, erythroid burst-forming units, and multilineage colony-forming units. As compared with mock-injected mice, administration of rhG-CSF alone (5 micro g/mouse/day) for 5 days significantly (P < or = 0.0001) increased WBC counts, CFC frequencies, and CFC absolute numbers by 2-, 13-, and 27-fold, respectively. As compared with control mice, the combined administration of Defibrotide (15 mg/mouse/day) and rhG-CSF significantly (P < or = 0.0001) increased WBC counts, frequencies and absolute numbers of CFCs by 4-, 38-, and 119-fold, respectively. As compared with rhG-CSF alone, administration of Defibrotide plus rhG-CSF resulted in a significant increase (P < or = 0.001) of the frequency of circulating long-term culture-initiating cells. In addition, transplantation of 2 x 10(5) rhG-CSF- or Defibrotide/rhG-CSF-mobilized mononuclear cells rescued 43% and 71% of recipient mice, respectively. Experiments of CFC homing performed in lethally irradiated or nonirradiated recipients showed that marrow homing of transplanted PBPCs was reduced by 3-fold in Defibrotide-treated animals as compared with mock-injected mice (P < or = 0.001), suggesting that the mobilizing effect of Defibrotide might be because of an effect on PBPC trafficking. In conclusion, our data demonstrate that Defibrotide synergizes with rhG-CSF and significantly increases the mobilization of a broad spectrum of PBPCs, including primitive and committed progenitor cells. These data might have relevant implications for autologous and allogeneic anticancer therapy in humans.

Peptide immunotherapy in allergic asthma generates IL-10–dependent immunological tolerance associated with linked epitope suppression

PubMed Central

Campbell, John D.; Buckland, Karen F.; McMillan, Sarah J.; Kearley, Jennifer; Oldfield, William L.G.; Stern, Lawrence J.; Grönlund, Hans; van Hage, Marianne; Reynolds, Catherine J.; Boyton, Rosemary J.; Cobbold, Stephen P.; Kay, A. Barry; Altmann, Daniel M.; Larché, Mark

2009-01-01

Treatment of patients with allergic asthma using low doses of peptides containing T cell epitopes from Fel d 1, the major cat allergen, reduces allergic sensitization and improves surrogate markers of disease. Here, we demonstrate a key immunological mechanism, linked epitope suppression, associated with this therapeutic effect. Treatment with selected epitopes from a single allergen resulted in suppression of responses to other (“linked”) epitopes within the same molecule. This phenomenon was induced after peptide immunotherapy in human asthmatic subjects and in a novel HLA-DR1 transgenic mouse model of asthma. Tracking of allergen-specific T cells using DR1 tetramers determined that suppression was associated with the induction of interleukin (IL)-10+ T cells that were more abundant than T cells specific for the single-treatment peptide and was reversed by anti–IL-10 receptor administration. Resolution of airway pathophysiology in this model was associated with reduced recruitment, proliferation, and effector function of allergen-specific Th2 cells. Our results provide, for the first time, in vivo evidence of linked epitope suppression and IL-10 induction in both human allergic disease and a mouse model designed to closely mimic peptide therapy in humans. PMID:19528258

Cloning and characterization of the rat free fatty acid receptor GPR120: in vivo effect of the natural ligand on GLP-1 secretion and proliferation of pancreatic beta cells.

PubMed

Tanaka, Toshiki; Yano, Takeaki; Adachi, Tetsuya; Koshimizu, Taka-aki; Hirasawa, Akira; Tsujimoto, Gozoh

2008-06-01

We have recently found that GPR120, which is abundantly expressed in intestine, functions as a receptor for unsaturated long-chain free fatty acids (FFAs) and that GPR120 stimulation promotes the secretion of glucagons-like peptide-1 (GLP-1) in the mouse (Hirasawa et al., Nat Med 11:90-94, 2005). In this study, we cloned and characterized rat GPR120 (rGPR120), and then we examined the in vivo effects of acute and long-term administration of the natural ligand alpha-linolenic acid (alpha-LA). The cloned rat GPR120 complimentary DNA had a seven transmembrane structure, and a homology comparison of human, mouse, and rat GPR120 revealed that the rat GPR120 (rGPR120) shares 85 and 98% sequence identity with the human and mouse GPR120 proteins, respectively. The tissue distribution and ligand properties of rGPR120 were similar to those of mouse GPR120. In addition, alpha-LA provoked a transient increase in [Ca2+]i levels in HEK293 cells expressing rGPR120. Furthermore, administration of alpha-LA to the rat increased plasma GLP-1 levels, and long-term administration of alpha-LA led to proliferation of pancreatic beta cells, probably because of the enhanced GLP-1 secretion. These results show that rat GPR120 is a G-protein-coupled receptor whose ligand is a free fatty acid, and it may play an important role in the FFA-associated physiological responses.

MEK-ERK inhibition potentiates WAY-600-induced anti-cancer efficiency in preclinical hepatocellular carcinoma (HCC) models

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Kaifeng, E-mail: kaifeng_wangdr@sina.com; Fan, Yaohua; Chen, Gongying

The search for novel anti-hepatocellular carcinoma (HCC) agents is important. Mammalian target of rapamycin (mTOR) hyper-activation plays a pivotal role in promoting HCC tumorigenesis and chemoresistance. The current preclinical study evaluated the potential anti-HCC activity by a potent mTOR kinase inhibitor, WAY-600. We showed that WAY-600 inhibited survival and proliferation of HCC cell lines (HepG2 and Huh7) and primary human HCC cells. Caspase-dependent apoptosis was activated by WAY-600 in above HCC cells. Reversely, caspase inhibitors largely attenuated WAY-600's lethality against HCC cells. At the signaling level, WAY-600 blocked mTOR complex 1/2 (mTORC1/2) assemble and activation, yet activated MEK-ERK pathway inmore » HCC cells. MEK-ERK inhibitors, PD-98059 and MEK-162, or MEK1/2 shRNA significantly potentiated WAY-600's cytotoxicity in HCC cells. Further studies showed that WAY-600 intraperitoneal (i.p.) administration in nude mice inhibited p-AKT Ser-473 and displayed significant anti-cancer activity against HepG2 xenografts. Remarkably, co-administration of MEK-162 further potentiated WAY-600's anti-HCC activity in vivo. These preclinical results demonstrate the potent anti-HCC activity by WAY-600, either alone or with MEK-ERK inhibitors. -- Highlights: •WAY-600 inhibits HCC cell survival and proliferation in vitro. •WAY-600 activates caspase-dependent apoptosis in HCC cells. •WAY-600 blocks mTORC1/2 activation, but activates MEK-ERK in HCC cells. •MEK-ERK inhibitors or MEK1/2 shRNA enhances WAY-600's cytotoxicity against HCC cells. •MEK-162 co-administration potentiates WAY-600-induced the anti-HepG2 tumor efficacy.« less

Enhancement of the blood compatibility of dialyzer membranes by the physical adsorption of human thrombomodulin (ART-123).

PubMed

Omichi, Masaaki; Matsusaki, Michiya; Kato, Shinya; Maruyama, Ikuro; Akashi, Mitsuru

2010-11-01

ART-123 is a recombinant soluble human thrombomodulin (hTM) with excellent anticoagulant activity. We focused on improving the blood compatibility of the polysulfone-polyvinylpyrrolidone dialyzer surface by the physical adsorption of ART-123 onto the surface. The blood compatibility of the dialyzer with the hTM adsorbed membrane was evaluated by measuring the differential pressure between the arterial and the venous pressures and by blood parameters during blood circulation. The hTM adsorbed dialyzer membrane inhibited blood clot formation without heparin administration due to the anticoagulant activity of hTM for over 4 h. The physically adsorbed hTM was stable during blood circulation, and it did not affect activated clotting time, which is significant drawback of heparin administration, and blood cell counts of RBC, WBC, or platelets. The physical adsorption of hTM onto the dialyzer membrane will be a simple and safe method to prevent blood coagulation during dialysis instead of heparin administration. © 2010 Wiley Periodicals, Inc.

In Vitro Pre-Clinical Validation of Suicide Gene Modified Anti-CD33 Redirected Chimeric Antigen Receptor T-Cells for Acute Myeloid Leukemia

PubMed Central

Minagawa, Kentaro; Jamil, Muhammad O.; AL-Obaidi, Mustafa; Pereboeva, Larisa; Salzman, Donna; Erba, Harry P.; Lamb, Lawrence S.; Bhatia, Ravi; Mineishi, Shin

2016-01-01

Background Approximately fifty percent of patients with acute myeloid leukemia can be cured with current therapeutic strategies which include, standard dose chemotherapy for patients at standard risk of relapse as assessed by cytogenetic and molecular analysis, or high-dose chemotherapy with allogeneic hematopoietic stem cell transplant for high-risk patients. Despite allogeneic hematopoietic stem cell transplant about 25% of patients still succumb to disease relapse, therefore, novel strategies are needed to improve the outcome of patients with acute myeloid leukemia. Methods and findings We developed an immunotherapeutic strategy targeting the CD33 myeloid antigen, expressed in ~ 85–90% of patients with acute myeloid leukemia, using chimeric antigen receptor redirected T-cells. Considering that administration of CAR T-cells has been associated with cytokine release syndrome and other potential off-tumor effects in patients, safety measures were here investigated and reported. We genetically modified human activated T-cells from healthy donors or patients with acute myeloid leukemia with retroviral supernatant encoding the inducible Caspase9 suicide gene, a ΔCD19 selectable marker, and a humanized third generation chimeric antigen receptor recognizing human CD33. ΔCD19 selected inducible Caspase9-CAR.CD33 T-cells had a 75±3.8% (average ± standard error of the mean) chimeric antigen receptor expression, were able to specifically lyse CD33+ targets in vitro, including freshly isolated leukemic blasts from patients, produce significant amount of tumor-necrosis-factor-alpha and interferon-gamma, express the CD107a degranulation marker, and proliferate upon antigen specific stimulation. Challenging ΔCD19 selected inducible Caspase9-CAR.CD33 T-cells with programmed-death-ligand-1 enriched leukemia blasts resulted in significant killing like observed for the programmed-death-ligand-1 negative leukemic blasts fraction. Since the administration of 10 nanomolar of a non-therapeutic dimerizer to activate the suicide gene resulted in the elimination of only 76.4±2.0% gene modified cells in vitro, we found that co-administration of the dimerizer with either the BCL-2 inhibitor ABT-199, the pan-BCL inhibitor ABT-737, or mafosfamide, resulted in an additive effect up to complete cell elimination. Conclusions This strategy could be investigated for the safety of CAR T-cell applications, and targeting CD33 could be used as a ‘bridge” therapy for patients coming to allogeneic hematopoietic stem cell transplant, as anti-leukemia activity from infusing CAR.CD33 T-cells has been demonstrated in an ongoing clinical trial. Albeit never performed in the clinical setting, our future plan is to investigate the utility of iC9-CAR.CD33 T-cells as part of the conditioning therapy for an allogeneic hematopoietic stem cell transplant for acute myeloid leukemia, together with other myelosuppressive agents, whilst the activation of the inducible Caspase9 suicide gene would grant elimination of the infused gene modified T-cells prior to stem cell infusion to reduce the risk of engraftment failure as the CD33 is also expressed on a proportion of the donor stem cell graft. PMID:27907031

Simultaneous Overexpression of Functional Human HO-1, E5NT and ENTPD1 Protects Murine Fibroblasts against TNF-α-Induced Injury In Vitro

PubMed Central

Cinti, Alessandro; De Giorgi, Marco; Chisci, Elisa; Arena, Claudia; Galimberti, Gloria; Farina, Laura; Bugarin, Cristina; Rivolta, Ilaria; Gaipa, Giuseppe; Smolenski, Ryszard Tom; Cerrito, Maria Grazia; Lavitrano, Marialuisa; Giovannoni, Roberto

2015-01-01

Several biomedical applications, such as xenotransplantation, require multiple genes simultaneously expressed in eukaryotic cells. Advances in genetic engineering technologies have led to the development of efficient polycistronic vectors based on the use of the 2A self-processing oligopeptide. The aim of this work was to evaluate the protective effects of the simultaneous expression of a novel combination of anti-inflammatory human genes, ENTPD1, E5NT and HO-1, in eukaryotic cells. We produced an F2A system-based multicistronic construct to express three human proteins in NIH3T3 cells exposed to an inflammatory stimulus represented by tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine which plays an important role during inflammation, cell proliferation, differentiation and apoptosis and in the inflammatory response during ischemia/reperfusion injury in several organ transplantation settings. The protective effects against TNF-α-induced cytotoxicity and cell death, mediated by HO-1, ENTPD1 and E5NT genes were better observed in cells expressing the combination of genes as compared to cells expressing each single gene and the effect was further improved by administrating enzymatic substrates of the human genes to the cells. Moreover, a gene expression analyses demonstrated that the expression of the three genes has a role in modulating key regulators of TNF-α signalling pathway, namely Nemo and Tnfaip3, that promoted pro-survival phenotype in TNF-α injured cells. These results could provide new insights in the research of protective mechanisms in transplantation settings. PMID:26513260

Chimeric antigen receptor–engineered T cells as oncolytic virus carriers

PubMed Central

VanSeggelen, Heather; Tantalo, Daniela GM; Afsahi, Arya; Hammill, Joanne A; Bramson, Jonathan L

2015-01-01

The use of engineered T cells in adoptive transfer therapies has shown significant promise in treating hematological cancers. However, successes treating solid tumors are much less prevalent. Oncolytic viruses (OVs) have the capacity to induce specific lysis of tumor cells and indirectly impact tumor growth via vascular shutdown. These viruses bear natural abilities to associate with lymphocytes upon systemic administration, but therapeutic doses must be very high in order to evade antibodies and other components of the immune system. As T cells readily circulate through the body, using these cells to deliver OVs directly to tumors may provide an ideal combination. Our studies demonstrate that loading chimeric antigen receptor–engineered T cells with low doses of virus does not impact receptor expression or function in either murine or human T cells. Engineered T cells can deposit virus onto a variety of tumor targets, which can enhance the tumoricidal activity of the combination treatment. This concept appears to be broadly applicable, as we observed similar results using murine or human T cells, loaded with either RNA or DNA viruses. Overall, loading of engineered T cells with OVs represents a novel combination therapy that may increase the efficacy of both treatments. PMID:27119109

Coenzyme Q10 Protects Human Endothelial Cells from β-Amyloid Uptake and Oxidative Stress-Induced Injury

PubMed Central

Durán-Prado, Mario; Frontiñán, Javier; Santiago-Mora, Raquel; Peinado, Juan Ramón; Parrado-Fernández, Cristina; Gómez-Almagro, María Victoria; Moreno, María; López-Domínguez, José Alberto; Villalba, José Manuel; Alcaín, Francisco J.

2014-01-01

Neuropathological symptoms of Alzheimer's disease appear in advances stages, once neuronal damage arises. Nevertheless, recent studies demonstrate that in early asymptomatic stages, ß-amyloid peptide damages the cerebral microvasculature through mechanisms that involve an increase in reactive oxygen species and calcium, which induces necrosis and apoptosis of endothelial cells, leading to cerebrovascular dysfunction. The goal of our work is to study the potential preventive effect of the lipophilic antioxidant coenzyme Q (CoQ) against ß-amyloid-induced damage on human endothelial cells. We analyzed the protective effect of CoQ against Aβ-induced injury in human umbilical vein endothelial cells (HUVECs) using fluorescence and confocal microscopy, biochemical techniques and RMN-based metabolomics. Our results show that CoQ pretreatment of HUVECs delayed Aβ incorporation into the plasma membrane and mitochondria. Moreover, CoQ reduced the influx of extracellular Ca2+, and Ca2+ release from mitochondria due to opening the mitochondrial transition pore after β-amyloid administration, in addition to decreasing O2 .− and H2O2 levels. Pretreatment with CoQ also prevented ß-amyloid-induced HUVECs necrosis and apoptosis, restored their ability to proliferate, migrate and form tube-like structures in vitro, which is mirrored by a restoration of the cell metabolic profile to control levels. CoQ protected endothelial cells from Aβ-induced injury at physiological concentrations in human plasma after oral CoQ supplementation and thus could be a promising molecule to protect endothelial cells against amyloid angiopathy. PMID:25272163

Evaluation of motor neuron differentiation potential of human umbilical cord blood- derived mesenchymal stem cells, in vitro.

PubMed

Yousefi, Behnam; Sanooghi, Davood; Faghihi, Faezeh; Joghataei, Mohammad Taghi; Latifi, Nourahmad

2017-04-01

Many people suffer from spinal cord injuries annually. These deficits usually threaten the quality of life of patients. As a postpartum medically waste product, human Umbilical Cord Blood (UCB) is a rich source of stem cells with self- renewal properties and neural differentiation capacity which made it useful in regenerative medicine. Since there is no report on potential of human umbilical cord blood-derived mesenchymal stem cells into motor neurons, we set out to evaluate the differentiation properties of these cells into motor neuron-like cells through administration of Retinoic Acid(RA), Sonic Hedgehog(Shh) and BDNF using a three- step in vitro procedure. The results were evaluated using Real-time PCR, Flowcytometry and Immunocytochemistry for two weeks. Our data showed that the cells changed into bipolar morphology and could express markers related to motor neuron; including Hb-9, Pax-6, Islet-1, NF-H, ChAT at the level of mRNA and protein. We could also quantitatively evaluate the expression of Islet-1, ChAT and NF-H at 7 and 14days post- induction using flowcytometry. It is concluded that human UCB-MSCs is potent to express motor neuron- related markers in the presence of RA, Shh and BDNF through a three- step protocol; thus it could be a suitable cell candidate for regeneration of motor neurons in spinal cord injuries. Copyright © 2017. Published by Elsevier B.V.

Effect of lactobacilli on paracellular permeability in the gut.

PubMed

Ahrne, Siv; Hagslatt, Marie-Louise Johansson

2011-01-01

Paracellular permeability is determined by the complex structures of junctions that are located between the epithelial cells. Already in 1996, it was shown that the human probiotic strain Lactobacillus plantarum 299v and the rat-originating strain Lactobacillus reuteri R2LC could reduce this permeability in a methotrexate-induced colitis model in the rat. Subsequently, many animal models and cell culture systems have shown indications that lactobacilli are able to counteract increased paracellular permeability evoked by cytokines, chemicals, infections, or stress. There have been few human studies focusing on the effect of lactobacilli on intestinal paracellular permeability but recently it has been shown that they could influence the tight junctions. More precisely, short-term administration of L. plantarum WCSF1 to healthy volunteers increased the relocation of occludin and ZO-1 into the tight junction area between duodenal epithelial cells.

Nanotoxicity of poly(n-butylcyano-acrylate) nanoparticles at the blood-brain barrier, in human whole blood and in vivo.

PubMed

Kolter, Marise; Ott, Melanie; Hauer, Christian; Reimold, Isolde; Fricker, Gert

2015-01-10

Therapy of diseases of the central nervous system is a major challenge since drugs have to overcome the blood-brain barrier (BBB). A powerful strategy to enhance cerebral drug concentration is administration of drug-loaded poly(n-butylcyano-acrylate) (PBCA) nanoparticles coated with polysorbate 80 (PS80). This study evaluates the toxicity of PBCA-nanoparticles at the BBB, representing the target organ, the inflammatory response in human whole blood, as the site of administration and in a rat model in vivo. PBCA-nanoparticles were prepared by a mini-emulsion method and characterized concerning size, surface charge, shape and PS80-adsorption. The influence on metabolic activity, cell viability and integrity of the BBB was analyzed in an in vitro model of the BBB. In ex vivo experiments in human whole blood the release of 12 inflammatory cytokines was investigated. In addition, the inflammatory response was studied in vivo in rats and complemented with the analysis of different organ toxicity parameters. PBCA-nanoparticles showed time- and concentration-dependent effects on metabolic activity, cell viability and BBB integrity. No cell death or loss of metabolic activity was observed for nanoparticle-concentrations ≤500μg/ml up to 3h of treatment. Within 12 tested inflammatory cytokines, only interleukin-8 displayed a significant release after nanoparticle exposure in human blood. No severe inflammatory processes or organ damages were identified in rats in vivo. Thus, PBCA-nanoparticles are a promising drug delivery system to overcome the BBB since they showed hardly any cytotoxic or inflammatory effect at therapeutic concentrations and incubation times. Copyright © 2014 Elsevier B.V. All rights reserved.

A Red Blood Cell Membrane-Camouflaged Nanoparticle Counteracts Streptolysin O-Mediated Virulence Phenotypes of Invasive Group A Streptococcus

PubMed Central

Escajadillo, Tamara; Olson, Joshua; Luk, Brian T.; Zhang, Liangfang; Nizet, Victor

2017-01-01

Group A Streptococcus (GAS), an important human-specific Gram-positive bacterial pathogen, is associated with a broad spectrum of disease, ranging from mild superficial infections such as pharyngitis and impetigo, to serious invasive infections including necrotizing fasciitis and streptococcal toxic shock syndrome. The GAS pore-forming streptolysin O (SLO) is a well characterized virulence factor produced by nearly all GAS clinical isolates. High level expression of SLO is epidemiologically linked to intercontinental dissemination of hypervirulent clonotypes and poor clinical outcomes. SLO can trigger macrophage and neutrophil cell death and/or the inactivation of immune cell functions, and promotes tissue injury and bacterial survival in animal models of infection. In the present work, we describe how the pharmacological presentation of red blood cell (RBC) derived biomimetic nanoparticles (“nanosponges”) can sequester SLO and block the ability of GAS to damage host cells, thereby preserving innate immune function and increasing bacterial clearance in vitro and in vivo. Nanosponge administration protected human neutrophils, macrophages, and keratinocytes against SLO-mediated cytotoxicity. This therapeutic intervention prevented SLO-induced macrophage apoptosis and increased neutrophil extracellular trap formation, allowing increased GAS killing by the respective phagocytic cell types. In a murine model of GAS necrotizing skin infection, local administration of the biomimetic nanosponges was associated with decreased lesion size and reduced bacterial colony-forming unit recovery. Utilization of a toxin decoy and capture platform that inactivates the secreted SLO before it contacts the host cell membrane, presents a novel virulence factor targeted strategy that could be a powerful adjunctive therapy in severe GAS infections where morbidity and mortality are high despite antibiotic treatment. PMID:28769806

Effect of doxorubicin, oxaliplatin, and methotrexate administration on the transcriptional activity of BCL-2 family gene members in stomach cancer cells.

PubMed

Florou, Dimitra; Patsis, Christos; Ardavanis, Alexandros; Scorilas, Andreas

2013-07-01

Defective apoptosis comprises the main reason for tumor aggressiveness and chemotherapy tolerance in solid neoplasias. Among the BCL-2 family members, whose mRNA or protein expression varies considerably in different human malignancies, BCL2L12 is the one for which we have recently shown its propitious prognostic value in gastric cancer. The purpose of the current work was to investigate the expression behavior of BCL2L12, BAX, and BCL-2 in human stomach adenocarcinoma cells following their exposure to anti-tumor substances. The 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide and trypan blue methods assessed the impact of doxorubicin, oxaliplatin and methotrexate on AGS cells' viability and growth. Following isolation from cells, total RNA was reverse-transcribed to cDNA. Quantification of target genes' expression was performed with real-time PCR using SYBR Green detection system. The relative changes in their mRNA levels between drug-exposed and untreated cells were calculated with the comparative Ct method (2(-ddCt)). All three drugs, as a result of their administration to AGS cancer cells for particular time intervals, provoked substantial fluctuations in the transcriptional levels of the apoptosis-related genes studied. While BAX was principally upregulated, striking similar were the notable changes regarding BCL-2 and BCL2L12 expression in our cellular system. Our findings indicate the growth suppressive effects of doxorubicin, oxaliplatin and methotrexate treatment on stomach carcinoma cells and the implication of BCL2L12, BAX, and BCL-2 expression profiles in the molecular signaling pathways triggered by chemotherapy.

Leptin promotes wound healing in the oral mucosa.

PubMed

Umeki, Hirochika; Tokuyama, Reiko; Ide, Shinji; Okubo, Mitsuru; Tadokoro, Susumu; Tezuka, Mitsuki; Tatehara, Seiko; Satomura, Kazuhito

2014-01-01

Leptin, a 16 kDa circulating anti-obesity hormone, exhibits many physiological properties. Recently, leptin was isolated from saliva; however, its function in the oral cavity is still unclear. In this study, we investigated the physiological role of leptin in the oral cavity by focusing on its effect on wound healing in the oral mucosa. Immunohistochemical analysis was used to examine the expression of the leptin receptor (Ob-R) in human/rabbit oral mucosa. To investigate the effect of leptin on wound healing in the oral mucosa, chemical wounds were created in rabbit oral mucosa, and leptin was topically administered to the wound. The process of wound repair was histologically observed and quantitatively analyzed by measuring the area of ulceration and the duration required for complete healing. The effect of leptin on the proliferation, differentiation and migration of human oral mucosal epithelial cells (RT7 cells) was investigated using crystal violet staining, reverse transcription polymerase chain reaction (RT-PCR) and a wound healing assay, respectively. Ob-R was expressed in spinous/granular cells in the epithelial tissue and vascular endothelial cells in the subepithelial connective tissue of the oral mucosa. Topical administration of leptin significantly promoted wound healing and shortened the duration required for complete healing. Histological analysis of gingival tissue beneath the ulceration showed a denser distribution of blood vessels in the leptin-treated group. Although the proliferation and differentiation of RT7 cells were not affected by leptin, the migration of these cells was accelerated in the presence of leptin. Topically administered leptin was shown to promote wound healing in the oral mucosa by accelerating epithelial cell migration and enhancing angiogenesis around the wounded area. These results strongly suggest that topical administration of leptin may be useful as a treatment to promote wound healing in the oral mucosa.

Administration of Interleukin-6 Stimulates Multilineage Hematopoiesis and Accelerates Recovery from Radiation-Induced Hematopoietic Depression

DTIC Science & Technology

1991-02-01

Htirano T, Kishimoto T, Nakahata T. Asano S: In vitro hernatopoietic growth factors. J1 Natl Cancer Inst 81: t370. 1989 expansion of the murine...a.i phas S. %’ink A. Billiau A. VanSnick 1: Identification of the nutohlbyrcmiatntrekn.Cllmuolt12. human Zh-kd protein, interferon beta ,, as a B... beta . B-cell %timulatory factor type 2 shares identity T. Takaku F. Akivama Y: In vivo effects of recombinant human with rnsanc~tc-derived

Effects of cellular origin on differentiation of human induced pluripotent stem cell–derived endothelial cells

PubMed Central

Zhao, Ming-Tao; Jahanbani, Fereshteh; Lee, Won Hee; Snyder, Michael P.

2016-01-01

Human induced pluripotent stem cells (iPSCs) can be derived from various types of somatic cells by transient overexpression of 4 Yamanaka factors (OCT4, SOX2, C-MYC, and KLF4). Patient-specific iPSC derivatives (e.g., neuronal, cardiac, hepatic, muscular, and endothelial cells [ECs]) hold great promise in drug discovery and regenerative medicine. In this study, we aimed to evaluate whether the cellular origin can affect the differentiation, in vivo behavior, and single-cell gene expression signatures of human iPSC–derived ECs. We derived human iPSCs from 3 types of somatic cells of the same individuals: fibroblasts (FB-iPSCs), ECs (EC-iPSCs), and cardiac progenitor cells (CPC-iPSCs). We then differentiated them into ECs by sequential administration of Activin, BMP4, bFGF, and VEGF. EC-iPSCs at early passage (10 < P < 20) showed higher EC differentiation propensity and gene expression of EC-specific markers (PECAM1 and NOS3) than FB-iPSCs and CPC-iPSCs. In vivo transplanted EC-iPSC–ECs were recovered with a higher percentage of CD31+ population and expressed higher EC-specific gene expression markers (PECAM1, KDR, and ICAM) as revealed by microfluidic single-cell quantitative PCR (qPCR). In vitro EC-iPSC–ECs maintained a higher CD31+ population than FB-iPSC–ECs and CPC-iPSC–ECs with long-term culturing and passaging. These results indicate that cellular origin may influence lineage differentiation propensity of human iPSCs; hence, the somatic memory carried by early passage iPSCs should be carefully considered before clinical translation. PMID:27398408

Oral or parenteral administration of replication-deficient adenoviruses expressing the measles virus haemagglutinin and fusion proteins: protective immune responses in rodents.

PubMed

Fooks, A R; Jeevarajah, D; Lee, J; Warnes, A; Niewiesk, S; ter Meulen, V; Stephenson, J R; Clegg, J C

1998-05-01

The genes encoding the measles virus (MV) haemagglutinin (H) and fusion (F) proteins were placed under the control of the human cytomegalovirus immediate early promoter in a replication-deficient adenovirus vector. Immunofluorescence and radioimmune precipitation demonstrated the synthesis of each protein and biological activity was confirmed by the detection of haemadsorption and fusion activities in infected cells. Oral as well as parenteral administration of the H-expressing recombinant adenovirus elicited a significant protective response in mice challenged with MV. While the F-expressing adenovirus failed to protect mice, cotton rats immunized with either the H- or F-expressing recombinant showed reduced MV replication in the lungs. Antibodies elicited in mice following immunization with either recombinant had no in vitro neutralizing activity, suggesting a protective mechanism involving a cell-mediated immune response. This study demonstrates the feasibility of using oral administration of adenovirus recombinants to induce protective responses to heterologous proteins.

Mycotoxins' activity at toxic and sub-toxic concentrations: differential cytotoxic and genotoxic effects of single and combined administration of sterigmatocystin, ochratoxin A and citrinin on the hepatocellular cancer cell line Hep3B.

PubMed

Anninou, Nikolia; Chatzaki, Ekaterini; Papachristou, Fotini; Pitiakoudis, Muichail; Simopoulos, Constantinos

2014-02-07

Food safety organizations indicate the likelihood of constant human and animal exposure to mycotoxin mixtures as a possible negative public health impact. Risk assessment demonstrates that certain mycotoxins of Aspergillus and Penicillium spp. are toxic and hold a significant genotoxic efficacy at nanomolar concentrations. The aim of the current study was to investigate the potential cytogenetic effects of sterigmatocystin (STER), ochratoxin A (OTA) and citrinin (CTN) alone or in combination, at pM to μΜ concentrations, on the human hepatocellular cancer cell line Hep3B. MTT reduction, mitotic divisions, cell cycle delays and sister chromatid exchange rates (SCE) were determined as endpoints of metabolic activity, cytotoxicity, cytostaticity, and genotoxicity, respectively. All mycotoxin treatments induce SCE rates from 10-12 M, while their cytotoxic and cytostatic potential varies. In PRI and MI assays, but not at MTT, STER alone or in combination with OTA + CTN appeared cytostatic and cytotoxic, even at 10-12 M, while CTN alone and all other combinations displayed substantial cellular survival inhibition in doses ≥ 10-8 M. Co-administration of STER + OTA or STER + CTN in concentrations ≤ 10-1 M, increased the MI and MTT activity, while it did not affect the PRI. Mycotoxin co-treatments revealed in general similar-to-additive or antagonistic genotoxic and cytotoxic effects. Our results for the first time describe that STER alone or in combination with OTA and/or CTN share a cytotoxic and cytogenetic potential even at picoMolar concentrations on human hepatoma cells in vitro.

B Cell Development in the Bone Marrow Is Regulated by Homeostatic Feedback Exerted by Mature B Cells

PubMed Central

Shahaf, Gitit; Zisman-Rozen, Simona; Benhamou, David; Melamed, Doron; Mehr, Ramit

2016-01-01

Cellular homeostasis in the B cell compartment is strictly imposed to balance cell production and cell loss. However, it is not clear whether B cell development in the bone marrow is an autonomous process or subjected to regulation by the peripheral B cell compartment. To specifically address this question, we used mice transgenic for human CD20, where effective depletion of B lineage cells is obtained upon administration of mouse anti-human CD20 antibodies, in the absence of any effect on other cell lineages and/or tissues. We followed the kinetics of B cell return to equilibrium by BrdU labeling and flow cytometry and analyzed the resulting data by mathematical modeling. Labeling was much faster in depleted mice. Compared to control mice, B cell-depleted mice exhibited a higher proliferation rate in the pro-/pre-B compartment, and higher cell death and lower differentiation in the immature B cell compartment. We validated the first result by analysis of the expression of Ki67, the nuclear protein expressed in proliferating cells, and the second using Annexin V staining. Collectively, our results suggest that B lymphopoiesis is subjected to homeostatic feedback mechanisms imposed by mature B cells in the peripheral compartment. PMID:27047488

Status quo of management of the human tissue banks in Taiwan.

PubMed

Chou, Ching-Pang; Chou, Szu-Cheng; Chen, Ying-Hua; Chen, Yu-Hsuan; Lee, Ming-Shin

2017-03-01

As the technologies associated with transplantation and biological tissue engineering continue to advance, human cells and tissues form an integral part to the practice of regenerative medicine. The patient's use of tissues entails the risk of introducing, transmitting and spreading communicable diseases. To prevent such risk and to ensure that the human organs, tissues and cells remain intact and functional after being handled and processed, the transplanted tissues must be subject to good management standards through all stages of collection, screening, processing, storage and distribution as the safety of the users is of the utmost importance. On February 2009, the government of Taiwan promulgated the Regulations for Administration on Human Organ Bank that requires all human tissues banks to adhere to the Good Tissue Practice for Human Organ, Tissue and Cell in terms of establishment and operation in order to cope with the international management trend and the development and management need of the domestic industry. Six years have passed since the law became effective. This article seeks to introduce the current management mechanism and status quo of management of human tissue banks in Taiwan. We also conducted statistical analysis of the data relating to the tissue banks to identify potential risks and the room for improvement. The study concludes that human tissue banks in Taiwan are on the right track with their management practice, leading to a state of steady development and progress.

Inhibitors of ORAI1 Prevent Cytosolic Calcium-Associated Injury of Human Pancreatic Acinar Cells and Acute Pancreatitis in 3 Mouse Models

PubMed Central

Wen, Li; Voronina, Svetlana; Javed, Muhammad A.; Awais, Muhammad; Szatmary, Peter; Latawiec, Diane; Chvanov, Michael; Collier, David; Huang, Wei; Barrett, John; Begg, Malcolm; Stauderman, Ken; Roos, Jack; Grigoryev, Sergey; Ramos, Stephanie; Rogers, Evan; Whitten, Jeff; Velicelebi, Gonul; Dunn, Michael; Tepikin, Alexei V.; Criddle, David N.; Sutton, Robert

2015-01-01

Background & Aims Sustained activation of the cytosolic calcium concentration induces injury to pancreatic acinar cells and necrosis. The calcium release–activated calcium modulator ORAI1 is the most abundant Ca2+ entry channel in pancreatic acinar cells; it sustains calcium overload in mice exposed to toxins that induce pancreatitis. We investigated the roles of ORAI1 in pancreatic acinar cell injury and the development of acute pancreatitis in mice. Methods Mouse and human acinar cells, as well as HEK 293 cells transfected to express human ORAI1 with human stromal interaction molecule 1, were hyperstimulated or incubated with human bile acid, thapsigargin, or cyclopiazonic acid to induce calcium entry. GSK-7975A or CM_128 were added to some cells, which were analyzed by confocal and video microscopy and patch clamp recordings. Acute pancreatitis was induced in C57BL/6J mice by ductal injection of taurolithocholic acid 3-sulfate or intravenous' administration of cerulein or ethanol and palmitoleic acid. Some mice then were given GSK-7975A or CM_128, which inhibit ORAI1, at different time points to assess local and systemic effects. Results GSK-7975A and CM_128 each separately inhibited toxin-induced activation of ORAI1 and/or activation of Ca2+ currents after Ca2+ release, in a concentration-dependent manner, in mouse and human pancreatic acinar cells (inhibition >90% of the levels observed in control cells). The ORAI1 inhibitors also prevented activation of the necrotic cell death pathway in mouse and human pancreatic acinar cells. GSK-7975A and CM_128 each inhibited all local and systemic features of acute pancreatitis in all 3 models, in dose- and time-dependent manners. The agents were significantly more effective, in a range of parameters, when given at 1 vs 6 hours after induction of pancreatitis. Conclusions Cytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis. ORAI1 inhibitors might be developed for the treatment of patients with pancreatitis. PMID:25917787

[Effect of BRL 25000 (clavulanic acid-amoxicillin) on bacterial flora in human feces].

PubMed

Motohiro, T; Tanaka, K; Koga, T; Shimada, Y; Tomita, N; Sakata, Y; Fujimoto, T; Nishiyama, T; Kuda, N; Ishimoto, K

1985-02-01

BRL 25000 (187.5 and 375 mg tablets), a formulation of CVA-K and AMPC in the ratio of 1:2, and AMPC (as control drug) were administered to healthy volunteers, aged 20 approximately 28 years and weighing 60 approximately 85 kg (68.8 kg, on average). Each drug was administered 3 times a day (after meals) for 5 days and the volunteers were separated into 3 groups of 4 subjects each. The effect on the fecal flora was studied before dosage, during administration (day 3 and 5) and day 3 and 5 after the administration course was completed. Studies were undertaken to isolate C. difficile on the last day of administration and 3 and 5 days after administration had ceased. Fecal concentrations and the susceptibility of the isolates to AMPC, CVA-K and BRL 25000 were measured. Side effects and laboratory findings were studied. The results obtained were as follows: 1. In BRL 25000 (187.5 mg X 3/day) group, the population of E. coli was on average, 1 X 10(6) approximately 9 X 10(6) cells/g feces before initiation of administration and it increased by 2 logarithms 3 and 5 days after initiation of administration. By 3 and 5 days after end of administration, the E. coli population was similar to the initial population. The population of Klebsiella sp. was 1 X 10(6) approximately 9 X 10(6) cells/g feces on average before commencement of dosage and it increased by 2 logarithms 3 days after initiation of administration but there was no consistent change in the Klebsiella sp. population thereafter. The Enterobacter sp., population was not consistent neither was the population of other Enterobacteriaceae. In total, the mean Enterobacteriaceae population was 1 X 10(7) approximately 9 X 10(7) cells/g feces before initiation of administration and increased by 2 logarithms 3 days after initiation of administration, and then returned to the initial level 5 days after end of administration. No consistent changes in population were noted for the other Gram-negative bacilli. The Staphylococcus sp. population was 1 X 10(6) approximately 9 X 10(6) cells/g feces on average before initiation of administration. This organism was detected in only 1 case 3 days after initiation of administration and in another 5 days after initiation of administration, thereafter, the population was similar to the initial population.(ABSTRACT TRUNCATED AT 400 WORDS)

Engineered Commensal Bacteria Reprogram Intestinal Cells Into Glucose-Responsive Insulin-Secreting Cells for the Treatment of Diabetes

PubMed Central

Duan, Franklin F.; Liu, Joy H.

2015-01-01

The inactive full-length form of GLP-1(1-37) stimulates conversion of both rat and human intestinal epithelial cells into insulin-secreting cells. We investigated whether oral administration of human commensal bacteria engineered to secrete GLP-1(1-37) could ameliorate hyperglycemia in a rat model of diabetes by reprogramming intestinal cells into glucose-responsive insulin-secreting cells. Diabetic rats were fed daily with human lactobacilli engineered to secrete GLP-1(1-37). Diabetic rats fed GLP-1–secreting bacteria showed significant increases in insulin levels and, additionally, were significantly more glucose tolerant than those fed the parent bacterial strain. These rats developed insulin-producing cells within the upper intestine in numbers sufficient to replace ∼25–33% of the insulin capacity of nondiabetic healthy rats. Intestinal tissues in rats with reprogrammed cells expressed MafA, PDX-1, and FoxA2. HNF-6 expression was observed only in crypt epithelia expressing insulin and not in epithelia located higher on the villous axis. Staining for other cell markers in rats treated with GLP-1(1-37)–secreting bacteria suggested that normal function was not inhibited by the close physical proximity of reprogrammed cells. These results provide evidence of the potential for a safe and effective nonabsorbed oral treatment for diabetes and support the concept of engineered commensal bacterial signaling to mediate enteric cell function in vivo. PMID:25626737

Human papillomavirus-mediated carcinogenesis and HPV-associated oral and oropharyngeal squamous cell carcinoma. Part 2: Human papillomavirus associated oral and oropharyngeal squamous cell carcinoma

PubMed Central

2010-01-01

Human papillomavirus (HPV) infection of the mouth and oropharynx can be acquired by a variety of sexual and social forms of transmission. HPV-16 genotype is present in many oral and oropharyngeal squamous cell carcinomata. It has an essential aetiologic role in the development of oropharyngeal squamous cell carcinoma in a subset of subjects who are typically younger, are more engaged with high-risk sexual behaviour, have higher HPV-16 serum antibody titer, use less tobacco and have better survival rates than in subjects with HPV-cytonegative oropharyngeal squamous cell carcinoma. In this subset of subjects the HPV-cytopositive carcinomatous cells have a distinct molecular profile. In contrast to HPV-cytopositive oropharyngeal squamous cell carcinoma, the causal association between HPV-16 and other high-risk HPV genotypes and squamous cell carcinoma of the oral mucosa is weak, and the nature of the association is unclear. It is likely that routine administration of HPV vaccination against high-risk HPV genotypes before the start of sexual activity will bring about a reduction in the incidence of HPV-mediated oral and oropharyngeal squamous cell carcinoma. This article focuses on aspects of HPV infection of the mouth and the oropharynx with emphasis on the link between HPV and squamous cell carcinoma, and on the limitations of the available diagnostic tests in identifying a cause-and-effect relationship of HPV with squamous cell carcinoma of the mouth and oropharynx. PMID:20633288

A Fusion Receptor as a Safety Switch, Detection, and Purification Biomarker for Adoptive Transferred T Cells.

PubMed

Wu, Xiuqi; Shi, Bizhi; Zhang, Jiqin; Shi, Zhimin; Di, Shengmeng; Fan, Minliang; Gao, Huiping; Wang, Hai; Gu, Jianren; Jiang, Hua; Li, Zonghai

2017-10-04

The incorporation of an endogenous safety switch represents a rational strategy for the control of toxicities following the administration of adoptive T cell therapies. An ideal safety switch should be capable of depleting the transferred T cells with minimal injury to normal tissues. We generated a fusion receptor by engineering a cryptic 806 epitope of human epidermal growth factor receptor (EGFR) into the N terminus of the full-length human folate receptor 1 (FOLR1), designated as FR806. The expression of FR806 allows transduced T cells to be targeted with CH12, a monoclonal antibody recognizing the 806 epitope, but not wild-type EGFR in healthy tissues. FR806, therefore, constitutes a specific cell-surface marker for the elimination of transduced T cells. We demonstrate that the antibody-drug conjugate (ADC) CH12-MMAF is efficiently internalized by FR806-expressing T cells and has the potential to eliminate them. Transfected T cells could, furthermore, be efficiently detected and purified using CH12 antibodies. In immuno-compromised mice, CH12-MMAF eliminated the majority of transferred T cells expressing FR806 and anti-CD19 chimeric antigen receptor (CAR). The selectivity for the 806 epitope and internalization capacity of FOLR1 makes FR806 an efficient safety switch, which may additionally be used as a detection and purification biomarker for human T cell immunotherapies. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Induction of cyclo-oxygenase-2 mRNA by prostaglandin E2 in human prostatic carcinoma cells

NASA Technical Reports Server (NTRS)

Tjandrawinata, R. R.; Dahiya, R.; Hughes-Fulford, M.

1997-01-01

Prostaglandins are synthesized from arachidonic acid by the enzyme cyclo-oxygenase. There are two isoforms of cyclooxygenases: COX-1 (a constitutive form) and COX-2 (an inducible form). COX-2 has recently been categorized as an immediate-early gene and is associated with cellular growth and differentiation. The purpose of this study was to investigate the effects of exogenous dimethylprostaglandin E2 (dmPGE2) on prostate cancer cell growth. Results of these experiments demonstrate that administration of dmPGE2 to growing PC-3 cells significantly increased cellular proliferation (as measured by the cell number), total DNA content and endogenous PGE2 concentration. DmPGE2 also increased the steady-state mRNA levels of its own inducible synthesizing enzyme, COX-2, as well as cellular growth to levels similar to those seen with fetal calf serum and phorbol ester. The same results were observed in other human cancer cell types, such as the androgen-dependent LNCaP cells, breast cancer MDA-MB-134 cells and human colorectal carcinoma DiFi cells. In PC-3 cells, the dmPGE2 regulation of the COX-2 mRNA levels was both time dependent, with maximum stimulation seen 2 h after addition, and dose dependent on dmPGE2 concentration, with maximum stimulation seen at 5 microg ml(-1). The non-steroidal anti-inflammatory drug flurbiprofen (5 microM), in the presence of exogenous dmPGE2, inhibited the up-regulation of COX-2 mRNA and PC-3 cell growth. Taken together, these data suggest that PGE2 has a specific role in the maintenance of human cancer cell growth and that the activation of COX-2 expression depends primarily upon newly synthesized PGE2, perhaps resulting from changes in local cellular PGE2 concentrations.

Preclinical characterization of AMG 330, a CD3/CD33-bispecific T-cell-engaging antibody with potential for treatment of acute myelogenous leukemia.

PubMed

Friedrich, Matthias; Henn, Anja; Raum, Tobias; Bajtus, Monika; Matthes, Katja; Hendrich, Larissa; Wahl, Joachim; Hoffmann, Patrick; Kischel, Roman; Kvesic, Majk; Slootstra, Jerry W; Baeuerle, Patrick A; Kufer, Peter; Rattel, Benno

2014-06-01

There is high demand for novel therapeutic options for patients with acute myelogenous leukemia (AML). One possible approach is the bispecific T-cell-engaging (BiTE, a registered trademark of Amgen) antibody AMG 330 with dual specificity for CD3 and the sialic acid-binding lectin CD33 (SIGLEC-3), which is frequently expressed on the surface of AML blasts and leukemic stem cells. AMG 330 binds with low nanomolar affinity to CD33 and CD3ε of both human and cynomolgus monkey origin. Eleven human AML cell lines expressing between 14,400 and 56,700 CD33 molecules per cell were all potently lysed with EC(50) values ranging between 0.4 pmol/L and 3 pmol/L (18-149 pg/mL) by previously resting, AMG 330-redirected T cells. Complete lysis was achieved after 40 hours of incubation. In the presence of AML cells, AMG 330 specifically induced expression of CD69 and CD25 as well as release of IFN-γ, TNF, interleukin (IL)-2, IL-10, and IL-6. Ex vivo, AMG 330 mediated autologous depletion of CD33-positive cells from cynomolgous monkey bone marrow aspirates. Soluble CD33 at concentrations found in bone marrow of patients with AML did not significantly affect activities of AMG 330. Neoexpression of CD33 on newly activated T cells was negligible as it was limited to 6% of T cells in only three out of ten human donors tested. Daily intravenous administration with as low as 0.002 mg/kg AMG 330 significantly prolonged survival of immunodeficient mice adoptively transferred with human MOLM-13 AML cells and human T cells. AMG 330 warrants further development as a potential therapy for AML. ©2014 American Association for Cancer Research.

Rebamipide increases the mucin-like glycoprotein production in corneal epithelial cells.

PubMed

Takeji, Yasuhiro; Urashima, Hiroki; Aoki, Akihiro; Shinohara, Hisashi

2012-06-01

Dry eye is a multifactorial disease of tears and the ocular surface due to tear deficiency or excessive tear evaporation. Tear film instability is due to a disturbance in ocular surface mucin leading to a dysfunction of mucin, resulting in dry eye. In this study, we examined the effect of rebamipide, an anti-ulcer agent, on glycoconjugate production, as an indicator of mucin-like glycoprotein in cultured corneal epithelial cells. Further, we investigated the effect of rebamipide on the gene expression of membrane-associated mucins. Confluent cultured human corneal epithelial cells were incubated with rebamipide for 24 h. The glycoconjugate content in the supernatant and the cell extracts was measured by wheat germ agglutinin-enzyme-linked lectin assay combined gel-filtration method. In the experiment on mucin gene expression, cultured human corneal epithelial cells were collected at 0, 3, 6, and 12 h after administration of rebamipide. Real-time quantitative polymerase chain reaction was used to analyze the quantity of MUC1, MUC 4, and MUC16 gene expression. Rebamipide significantly increased the glycoconjugate contents in the supernatant and cell extract. In the mucin gene expression in the cells, rebamipide increased MUC1 and MUC4 gene expression, but did not increase MUC16 gene expression. Rebamipide promoted glycoconjugate, which has a property as a mucin-like glycoprotein, in human corneal epithelial cells. The increased production was mediated by MUC1 and MUC4 gene expression.

Edaravone protects rats and human pulmonary alveolar epithelial cells against hyperoxia injury: heme oxygenase-1 and PI3K/Akt pathway may be involved.

PubMed

Cao, Huifang; Feng, Ying; Ning, Yunye; Zhang, Zinan; Li, Weihao; Li, Qiang

2015-01-01

Hyperoxic acute lung injury (HALI) is a clinical syndrome as a result of prolonged supplement of high concentrations of oxygen. As yet, no specific treatment is available for HALI. The present study aims to investigate the effects of edaravone on hyperoxia-induced oxidative injury and the underlying mechanism. We treated rats and human pulmonary alveolar epithelial cells with hyperoxia and different concentration of edaravone, then examined the effects of edaravone on cell viability, cell injury and two oxidative products. The roles of heme oxygenase-1 (HO-1) and PI3K/Akt pathway were explored using Western blot and corresponding inhibitors. The results showed that edaravone reduced lung biochemical alterations induced by hyperoxia and mortality of rats, dose-dependently alleviated cell mortality, cell injury, and peroxidation of cellular lipid and DNA oxidative damage. It upregulated cellular HO-1 expression and activity, which was reversed by PI3K/Akt pathway inhibition. The administration of zinc protoporphyrin-IX, a HO-1 inhibitor, and LY249002, a PI3K/Akt pathway inhibitor, abolished the protective effects of edaravone in cells. This study indicates that edaravone protects rats and human pulmonary alveolar epithelial cells against hyperoxia-induced injury and the antioxidant effect may be related to upregulation of HO-1, which is regulated by PI3K/Akt pathway.

Dissecting the mechanism of histone deacetylase inhibitors to enhance the activity of zinc finger nucleases delivered by integrase-defective lentiviral vectors.

PubMed

Joglekar, Alok V; Stein, Libby; Ho, Michelle; Hoban, Megan D; Hollis, Roger P; Kohn, Donald B

2014-07-01

Integrase-defective lentiviral vectors (IDLVs) have been of limited success in the delivery of zinc finger nucleases (ZFNs) to human cells, due to low expression. A reason for reduced gene expression has been proposed to involve the epigenetic silencing of vector genomes, carried out primarily by histone deacetylases (HDACs). In this study, we tested valproic acid (VPA), a known HDAC inhibitor (HDACi), for its ability to increase transgene expression from IDLVs, especially in the context of ZFN delivery. Using ZFNs targeting the human adenosine deaminase (ADA) gene in K562 cells, we demonstrated that treatment with VPA enhanced ZFN expression by up to 3-fold, resulting in improved allelic disruption at the ADA locus. Furthermore, three other U.S. Food and Drug Administration-approved HDACis (vorinostat, givinostat, and trichostatin-A) exhibited a similar effect on the activity of ZFN-IDLVs in K562 cells. In primary human CD34(+) cells, VPA- and vorinostat-treated cells showed higher levels of expression of both green fluorescent protein (GFP) as well as ZFNs from IDLVs. A major mechanism for the effects of HDAC inhibitors on improving expression was from their modulation of the cell cycle, and the influence of heterochromatinization was determined to be a lesser contributing factor.

Intestinal Cell Proliferation and Senescence Are Regulated by Receptor Guanylyl Cyclase C and p21*

PubMed Central

Basu, Nirmalya; Saha, Sayanti; Khan, Imran; Ramachandra, Subbaraya G.; Visweswariah, Sandhya S.

2014-01-01

Guanylyl cyclase C (GC-C) is expressed in intestinal epithelial cells and serves as the receptor for bacterial heat-stable enterotoxin (ST) peptides and the guanylin family of gastrointestinal hormones. Activation of GC-C elevates intracellular cGMP, which modulates intestinal fluid-ion homeostasis and differentiation of enterocytes along the crypt-villus axis. GC-C activity can regulate colonic cell proliferation by inducing cell cycle arrest, and mice lacking GC-C display increased cell proliferation in colonic crypts. Activation of GC-C by administration of ST to wild type, but not Gucy2c−/−, mice resulted in a reduction in carcinogen-induced aberrant crypt foci formation. In p53-deficient human colorectal carcinoma cells, ST led to a transcriptional up-regulation of p21, the cell cycle inhibitor, via activation of the cGMP-responsive kinase PKGII and p38 MAPK. Prolonged treatment of human colonic carcinoma cells with ST led to nuclear accumulation of p21, resulting in cellular senescence and reduced tumorigenic potential. Our results, therefore, identify downstream effectors for GC-C that contribute to regulating intestinal cell proliferation. Thus, genomic responses to a bacterial toxin can influence intestinal neoplasia and senescence. PMID:24217248

Intestinal lamina propria dendritic cells maintain T cell homeostasis but do not affect commensalism

PubMed Central

Welty, Nathan E.; Staley, Christopher; Ghilardi, Nico; Sadowsky, Michael J.; Igyártó, Botond Z.

2013-01-01

Dendritic cells (DCs) in the intestinal lamina propria (LP) are composed of two CD103+ subsets that differ in CD11b expression. We report here that Langerin is expressed by human LP DCs and that transgenic human langerin drives expression in CD103+CD11b+ LP DCs in mice. This subset was ablated in huLangerin-DTA mice, resulting in reduced LP Th17 cells without affecting Th1 or T reg cells. Notably, cognate DC–T cell interactions were not required for Th17 development, as this response was intact in huLangerin-Cre I-Aβfl/fl mice. In contrast, responses to intestinal infection or flagellin administration were unaffected by the absence of CD103+CD11b+ DCs. huLangerin-DTA x BatF3−/− mice lacked both CD103+ LP DC subsets, resulting in defective gut homing and fewer LP T reg cells. Despite these defects in LP DCs and resident T cells, we did not observe alterations of intestinal microbial communities. Thus, CD103+ LP DC subsets control T cell homeostasis through both nonredundant and overlapping mechanisms. PMID:24019552

Differential effects of Denileukin Diftitox IL-2 immunotoxin on NK and regulatory T cells in non-human primates

PubMed Central

Yamada, Yohei; Aoyama, Akihiro; Tocco, Georges; Boskovic, Svjetlan; Nadazdin, Ognjenka; Alessandrini, Alessandro; Madsen, Joren C.; Cosimi, A. Benedict; Benichou, Gilles; Kawai, Tatsuo

2012-01-01

Denileukin Diftitox (DD), a fusion protein comprised of IL-2 and diphtheria toxin was initially expected to enhance anti-tumor immunity by selectively eliminating regulatory T cells (Tregs) displaying the high affinity IL-2R (α-β-γ trimers). While DD has been shown to deplete some Tregs in primates, its effects on NK cells (CD16+CD8+NKG2A+CD3−), which constitutively express the intermediate affinity IL-2R (β-γ dimers) and play a critical role in anti-tumor immunity, are still unknown. To address this question, cynomolgus monkeys were injected intravenously with two different doses of DD (8 or 18 μg/Kg). This treatment resulted in a rapid but short-term reduction in detectable peripheral blood resting Tregs (R-Tregs: CD4+CD45RA+Foxp3+) and a transient increase in the number of activated Tregs (A-Tregs: CD4+CD45RA−Foxp3high) followed by their partial depletion (50–60%). On the other hand, all NK cells were deleted immediately and durably after DD administration. This difference was not due to a higher binding or internalization of DD by NK cells as compared to Tregs. Co-administration of DD with IL-15, which binds to IL-2Rβ-γ, abrogated DD-induced NK cell deletion in vitro and in vivo while it did not affect Tregs elimination. Taken together, these results show that DD exerts a potent cytotoxic effect on NK cells, a phenomenon which might impair its anti-tumoral properties. However, co-administration of IL-15 with DD could alleviate this problem by selectively protecting potentially oncolytic NK cells while allowing the depletion of immunosuppressive regulatory T cells in cancer patients. PMID:22586034

The opioid growth factor (OGF) and low dose naltrexone (LDN) suppress human ovarian cancer progression in mice.

PubMed

Donahue, Renee N; McLaughlin, Patricia J; Zagon, Ian S

2011-08-01

The opioid growth factor (OGF) and its receptor, OGFr, serve as a tonically active inhibitory axis regulating cell proliferation in normal cells and a variety of cancers, including human ovarian cancer. Blockade of OGF and OGFr with the nonselective opioid receptor antagonist naltrexone (NTX) upregulates expression of OGF and OGFr. Administration of a low dosage of NTX (LDN) blocks endogenous opioids from opioid receptors for a short period of time (4-6 h) each day, providing a window of 18-20 h for the upregulated opioids and receptors to interact. The present study investigated the repercussions of upregulating the OGF-OGFr axis by treatment with OGF or LDN on human ovarian tumorigenesis in vivo. Female nude mice were transplanted intraperitoneally with SKOV-3 human ovarian cancer cells and treated on a daily basis with OGF (10 mg/kg), LDN (0.1 mg/kg), or an equivalent volume of vehicle (saline). Tumor burden, as well as DNA synthesis, apoptosis, and angiogenesis was assessed in tumor tissue following 40 days of treatment. OGF and LDN markedly reduced ovarian tumor burden (tumor nodule number and weight). The mechanism of action was targeted to an inhibition of tumor cell proliferation and angiogenesis; no changes in cell survival were noted. This study shows that a native opioid pathway can suppress human ovarian cancer in a xenograft model, and provides novel non-toxic therapies for the treatment of this lethal neoplasia. Copyright © 2011 Elsevier Inc. All rights reserved.

In Vivo Imaging of the Human Retinal Pigment Epithelial Mosaic Using Adaptive Optics Enhanced Indocyanine Green Ophthalmoscopy

PubMed Central

Tam, Johnny; Liu, Jianfei; Dubra, Alfredo; Fariss, Robert

2016-01-01

Purpose The purpose of this study was to establish that retinal pigment epithelial (RPE) cells take up indocyanine green (ICG) dye following systemic injection and that adaptive optics enhanced indocyanine green ophthalmoscopy (AO-ICG) enables direct visualization of the RPE mosaic in the living human eye. Methods A customized adaptive optics scanning light ophthalmoscope (AOSLO) was used to acquire high-resolution retinal fluorescence images of residual ICG dye in human subjects after intravenous injection at the standard clinical dose. Simultaneously, multimodal AOSLO images were also acquired, which included confocal reflectance, nonconfocal split detection, and darkfield. Imaging was performed in 6 eyes of three healthy subjects with no history of ocular or systemic diseases. In addition, histologic studies in mice were carried out. Results The AO-ICG channel successfully resolved individual RPE cells in human subjects at various time points, including 20 minutes and 2 hours after dye administration. Adaptive optics-ICG images of RPE revealed detail which could be correlated with AO dark-field images of the same cells. Interestingly, there was a marked heterogeneity in the fluorescence of individual RPE cells. Confirmatory histologic studies in mice corroborated the specific uptake of ICG by the RPE layer at a late time point after systemic ICG injection. Conclusions Adaptive optics-enhanced imaging of ICG dye provides a novel way to visualize and assess the RPE mosaic in the living human eye alongside images of the overlying photoreceptors and other cells. PMID:27564519

In Vivo Imaging of the Human Retinal Pigment Epithelial Mosaic Using Adaptive Optics Enhanced Indocyanine Green Ophthalmoscopy.

PubMed

Tam, Johnny; Liu, Jianfei; Dubra, Alfredo; Fariss, Robert

2016-08-01

The purpose of this study was to establish that retinal pigment epithelial (RPE) cells take up indocyanine green (ICG) dye following systemic injection and that adaptive optics enhanced indocyanine green ophthalmoscopy (AO-ICG) enables direct visualization of the RPE mosaic in the living human eye. A customized adaptive optics scanning light ophthalmoscope (AOSLO) was used to acquire high-resolution retinal fluorescence images of residual ICG dye in human subjects after intravenous injection at the standard clinical dose. Simultaneously, multimodal AOSLO images were also acquired, which included confocal reflectance, nonconfocal split detection, and darkfield. Imaging was performed in 6 eyes of three healthy subjects with no history of ocular or systemic diseases. In addition, histologic studies in mice were carried out. The AO-ICG channel successfully resolved individual RPE cells in human subjects at various time points, including 20 minutes and 2 hours after dye administration. Adaptive optics-ICG images of RPE revealed detail which could be correlated with AO dark-field images of the same cells. Interestingly, there was a marked heterogeneity in the fluorescence of individual RPE cells. Confirmatory histologic studies in mice corroborated the specific uptake of ICG by the RPE layer at a late time point after systemic ICG injection. Adaptive optics-enhanced imaging of ICG dye provides a novel way to visualize and assess the RPE mosaic in the living human eye alongside images of the overlying photoreceptors and other cells.

Enteric nervous system abnormalities are present in human necrotizing enterocolitis: potential neurotransplantation therapy

PubMed Central

2013-01-01

Introduction Intestinal dysmotility following human necrotizing enterocolitis suggests that the enteric nervous system is injured during the disease. We examined human intestinal specimens to characterize the enteric nervous system injury that occurs in necrotizing enterocolitis, and then used an animal model of experimental necrotizing enterocolitis to determine whether transplantation of neural stem cells can protect the enteric nervous system from injury. Methods Human intestinal specimens resected from patients with necrotizing enterocolitis (n = 18), from control patients with bowel atresia (n = 8), and from necrotizing enterocolitis and control patients undergoing stoma closure several months later (n = 14 and n = 6 respectively) were subjected to histologic examination, immunohistochemistry, and real-time reverse-transcription polymerase chain reaction to examine the myenteric plexus structure and neurotransmitter expression. In addition, experimental necrotizing enterocolitis was induced in newborn rat pups and neurotransplantation was performed by administration of fluorescently labeled neural stem cells, with subsequent visualization of transplanted cells and determination of intestinal integrity and intestinal motility. Results There was significant enteric nervous system damage with increased enteric nervous system apoptosis, and decreased neuronal nitric oxide synthase expression in myenteric ganglia from human intestine resected for necrotizing enterocolitis compared with control intestine. Structural and functional abnormalities persisted months later at the time of stoma closure. Similar abnormalities were identified in rat pups exposed to experimental necrotizing enterocolitis. Pups receiving neural stem cell transplantation had improved enteric nervous system and intestinal integrity, differentiation of transplanted neural stem cells into functional neurons, significantly improved intestinal transit, and significantly decreased mortality compared with control pups. Conclusions Significant injury to the enteric nervous system occurs in both human and experimental necrotizing enterocolitis. Neural stem cell transplantation may represent a novel future therapy for patients with necrotizing enterocolitis. PMID:24423414

Changes in insulin-like growth factor-binding protein-3 messenger ribonucleic acid in endothelial cells of the human corpus luteum: a possible role in luteal development and rescue.

PubMed

Fraser, H M; Lunn, S F; Kim, H; Duncan, W C; Rodger, F E; Illingworth, P J; Erickson, G F

2000-04-01

In the human menstrual cycle, extensive angiogenesis accompanies luteinization; and the process is physiologically important for corpus luteum (CL) function. During luteolysis, the vasculature collapses, and the endothelial cells die. In a conceptual cycle, the CL persists both functionally and structurally beyond the luteoplacental shift. Although luteal rescue is not associated with increased angiogenesis, endothelial survival is extended. Despite the central role of the luteal vasculature in fertility, the mechanisms regulating its development and demise are poorly understood. There is increasing evidence that insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) may be important effectors of luteal function. Here, we have found that IGFBP-3 messenger RNA is expressed in the endothelium of the human CL and that the levels of message change during luteal development and rescue by human CG. The signal was strong during the early luteal phase, but it showed significant reduction during the mid- and late luteal phases. Interestingly, administration of human CG caused a marked increase in the levels of IGFBP-3 messenger RNA in luteal endothelial cells that was comparable to that observed during the early luteal phase. We conclude that endothelial cell IGFBP-3 expression is a physiological property of the CL of menstruation and pregnancy. These observations raise the intriguing possibility that the regulated expression of endothelial IGFBP-3 may play a role in controlling angiogenesis and cell responses in the human CL by autocrine/paracrine mechanisms.

Phenotypic and Functional Properties of Human Steady State CD14+ and CD1a+ Antigen Presenting Cells and Epidermal Langerhans Cells.

PubMed

Fehres, Cynthia M; Bruijns, Sven C M; Sotthewes, Brigit N; Kalay, Hakan; Schaffer, Lana; Head, Steven R; de Gruijl, Tanja D; Garcia-Vallejo, Juan J; van Kooyk, Yvette

2015-01-01

Cutaneous antigen presenting cells (APCs) are critical for the induction and regulation of skin immune responses. The human skin contains phenotypically and functionally distinct APCs subsets that are present at two separated locations. While CD1ahigh LCs form a dense network in the epidermis, the CD14+ and CD1a+ APCs reside in the dermal compartment. A better understanding of the biology of human skin APC subsets is necessary for the improvement of vaccine strategies that use the skin as administration route. In particular, progress in the characterization of uptake and activatory receptors will certainly improve APC-targeting strategies in vaccination. Here we performed a detailed analysis of the expression and function of glycan-binding and pattern-recognition receptors in skin APC subsets. The results demonstrate that under steady state conditions human CD1a+ dermal dendritic cells (DCs) were phenotypically most mature as measured by the expression of CD83 and CD86, whereas the CD14+ cells showed a higher expression of the CLRs DC-SIGN, mannose receptor and DCIR and had potent antigen uptake capacity. Furthermore, steady state LCs showed superior antigen cross-presentation as compared to the dermal APC subsets. Our results also demonstrate that the TLR3 ligand polyribosinic-polyribocytidylic acid (pI:C) was the most potent stimulator of cytokine production by both LCs and dDCs. These studies warrant further exploration of human CD1a+ dDCs and LCs as target cells for cancer vaccination to induce anti-tumor immune responses.

Consistency of the Proteome in Primary Human Keratinocytes With Respect to Gender, Age, and Skin Localization*

PubMed Central

Sprenger, Adrian; Weber, Sebastian; Zarai, Mostafa; Engelke, Rudolf; Nascimento, Juliana M.; Gretzmeier, Christine; Hilpert, Martin; Boerries, Melanie; Has, Cristina; Busch, Hauke; Bruckner-Tuderman, Leena; Dengjel, Jörn

2013-01-01

Keratinocytes account for 95% of all cells of the epidermis, the stratified squamous epithelium forming the outer layer of the skin, in which a significant number of skin diseases takes root. Immortalized keratinocyte cell lines are often used as research model systems providing standardized, reproducible, and homogenous biological material. Apart from that, primary human keratinocytes are frequently used for medical studies because the skin provides an important route for drug administration and is readily accessible for biopsies. However, comparability of these cell systems is not known. Cell lines may undergo phenotypic shifts and may differ from the in vivo situation in important aspects. Primary cells, on the other hand, may vary in biological functions depending on gender and age of the donor and localization of the biopsy specimen. Here we employed metabolic labeling in combination with quantitative mass spectrometry-based proteomics to assess A431 and HaCaT cell lines for their suitability as model systems. Compared with cell lines, comprehensive profiling of the primary human keratinocyte proteome with respect to gender, age, and skin localization identified an unexpected high proteomic consistency. The data were analyzed by an improved ontology enrichment analysis workflow designed for the study of global proteomics experiments. It enables a quick, comprehensive and unbiased overview of altered biological phenomena and links experimental data to literature. We guide through our workflow, point out its advantages compared with other methods and apply it to visualize differences of cell lines compared with primary human keratinocytes. PMID:23722187

Breast cancer instructs dendritic cells to prime interleukin 13–secreting CD4+ T cells that facilitate tumor development

PubMed Central

Aspord, Caroline; Pedroza-Gonzalez, Alexander; Gallegos, Mike; Tindle, Sasha; Burton, Elizabeth C.; Su, Dan; Marches, Florentina; Banchereau, Jacques; Palucka, A. Karolina

2007-01-01

We previously reported (Bell, D., P. Chomarat, D. Broyles, G. Netto, G.M. Harb, S. Lebecque, J. Valladeau, J. Davoust, K.A. Palucka, and J. Banchereau. 1999. J. Exp. Med. 190: 1417–1426) that breast cancer tumors are infiltrated with mature dendritic cells (DCs), which cluster with CD4+ T cells. We now show that CD4+ T cells infiltrating breast cancer tumors secrete type 1 (interferon γ) as well as high levels of type 2 (interleukin [IL] 4 and IL-13) cytokines. Immunofluorescence staining of tissue sections revealed intense IL-13 staining on breast cancer cells. The expression of phosphorylated signal transducer and activator of transcription 6 in breast cancer cells suggests that IL-13 actually delivers signals to cancer cells. To determine the link between breast cancer, DCs, and CD4+ T cells, we implanted human breast cancer cell lines in nonobese diabetic/LtSz-scid/scid β2 microglobulin–deficient mice engrafted with human CD34+ hematopoietic progenitor cells and autologous T cells. There, CD4+ T cells promote early tumor development. This is dependent on DCs and can be partially prevented by administration of IL-13 antagonists. Thus, breast cancer targets DCs to facilitate its development. PMID:17438063

Human cord blood-derived platelet lysate enhances the therapeutic activity of adipose-derived mesenchymal stromal cells isolated from Crohn's disease patients in a mouse model of colitis.

PubMed

Forte, Dorian; Ciciarello, Marilena; Valerii, Maria Chiara; De Fazio, Luigia; Cavazza, Elena; Giordano, Rosaria; Parazzi, Valentina; Lazzari, Lorenza; Laureti, Silvio; Rizzello, Fernando; Cavo, Michele; Curti, Antonio; Lemoli, Roberto M; Spisni, Enzo; Catani, Lucia

2015-09-09

Due to their immunomodulatory properties, mesenchymal stromal cells (MSCs) have been used for auto-immune disease treatment. Crohn disease (CD) and ulcerative colitis are two major inflammatory bowel diseases (IBDs), resulting from pathological immune responses to environmental or microbial antigens. Preclinical and clinical studies have suggested that MSC-based cellular therapy hold promising potential for IBD treatment. However, open issues include the selection of the proper cell dose, the source and the optimal route of administration of MSCs for more effective results. Platelet lysate has gained clinical interest due to its efficacy in accelerating wound healing. Thus, we propose to combine the administration of MSCs with a human umbilical cord blood-derived platelet lysate (hCBPL) as a novel strategy to improve MSC-based therapy for IBD resolution. Colitis was induced in 8-week-old C57BL/6J mice by daily oral administration of dextran sulphate sodium (DSS) (1.5 % w/v in tap water) for 9 days. MSCs were isolated from adipose tissue of CD patients (adCD-MSCs), expanded in proliferation medium, resuspended in hCBPL or PBS and administrated via enema for three times (1 × 10(6) cells/mouse/time) every other day starting on day +7 from DSS induction. The colitis evolution was evaluated by daily monitoring of body weight, stool consistency and bleeding. Histopathological analysis was performed. Inflammatory cytokine plasma levels were determined. adCD-MSCs stained with lipophilic membrane dye Nile Red, were injected in DSS mice as described above. Colon section of mice sacrificed 24 hours after last cell administration, were analyzed by confocal microscopy. We found that adCD-MSCs could be easily isolated and expanded from CD patients. Upon injection, adCD-MSCs exerted a therapeutic effect on DSS-induced colitis. Moreover, hCBPL increased adCD-MSCs efficacy by significantly reducing colitis scores, extension of the colon inflamed area and plasma levels of inflammatory mediators. Finally, Nile Red staining of MSCs is very efficient, stable and does not impair their vitality and function. Nile Red-labelling was clearly detected in the colitic area of adCD-MSCs injected mice and it was significantly brighter in the colon sections of mice that had received adCD-MSCs/hCBPL. In summary, with this study we propose a novel and promising adCD-MSC/hCBPL-based therapy for refractory IBDs.

A novel functional site of extracellular matrix metalloproteinase inducer (EMMPRIN) that limits the migration of human uterine cervical carcinoma cells.

PubMed

Sato, Takashi; Watanabe, Mami; Hashimoto, Kei; Ota, Tomoko; Akimoto, Noriko; Imada, Keisuke; Nomizu, Motoyoshi; Ito, Akira

2012-01-01

EMMPRIN (extracellular matrix metalloproteinase inducer)/CD147, a membrane-bound glycoprotein with two extracellular loop domains (termed loops I and II), progresses tumor invasion and metastasis by increasing the production of matrix metalloproteinase (MMP) in peritumoral stoma cells. EMMPRIN has also been associated with the control of migration activity in some tumor cells, but little is known about how EMMPRIN regulates tumor cell migration. In the present study, EMMPRIN siRNA suppressed the gene expression and production of EMMPRIN in human uterine cervical carcinoma SKG-II cells. An in vitro scratch wound assay showed enhancement of migration of EMMPRIN-knockdown SKG-II cells. In addition, the SKG-II cell migration was augmented by adding an E. coli-expressed human EMMPRIN mutant with two extracellular loop domains (eEMP-I/II), which bound to the cell surface of SKG-II cells. However, eEMP-I/II suppressed the native EMMPRIN-mediated augmentation of proMMP-1/procollagenase-1 production in a co-culture of the SKG-II cells and human uterine cervical fibroblasts, indicating that the augmentation of SKG-II cell migration resulted from the interference of native EMMPRIN functions by eEMP-I/II on the cell surface. Furthermore, a systematic peptide screening method using nine synthetic EMMPRIN peptides coding the loop I and II domains (termed EM1-9) revealed that EM9 (170HIENLNMEADPGQYR184) facilitated SKG-II cell migration. Moreover, SKG-II cell migration was enhanced by administration of an antibody against EM9, but not EM1 which is a crucial site for the MMP inducible activity of EMMPRIN. Therefore, these results provide novel evidence that EMMPRIN on the cell surface limits the cell migration of human uterine cervical carcinoma cells through 170HIENLNMEADPGQYR184 in the loop II domain. Finally, these results should provide an increased understanding of the functions of EMMPRIN in malignant cervical carcinoma cells, and could contribute to the development of clinical strategies for cervical cancer therapy.

A monoclonal antibody against KCNK9 K(+) channel extracellular domain inhibits tumour growth and metastasis.

PubMed

Sun, Han; Luo, Liqun; Lal, Bachchu; Ma, Xinrong; Chen, Lieping; Hann, Christine L; Fulton, Amy M; Leahy, Daniel J; Laterra, John; Li, Min

2016-02-04

Two-pore domain potassium (K2P) channels act to maintain cell resting membrane potential--a prerequisite for many biological processes. KCNK9, a member of K2P family, is implicated in cancer, owing to its overexpression in human tumours and its ability to promote neoplastic cell survival and growth. However, KCNK9's underlying contributions to malignancy remain elusive due to the absence of specific modulators. Here we describe the development of monoclonal antibodies against the KCNK9 extracellular domain and their functional effects. We show that one antibody (Y4) with the highest affinity binding induces channel internalization. The addition of Y4 to KCNK9-expressing carcinoma cells reduces cell viability and increases cell death. Systemic administration of Y4 effectively inhibits growth of human lung cancer xenografts and murine breast cancer metastasis in mice. Evidence for Y4-mediated carcinoma cell autonomous and immune-dependent cytotoxicity is presented. Our study reveals that antibody-based KCNK9 targeting is a promising therapeutic strategy in KCNK9-expressing malignancies.

F-box protein FBXW7 inhibits cancer metastasis in a non-cell-autonomous manner

PubMed Central

Yumimoto, Kanae; Akiyoshi, Sayuri; Ueo, Hiroki; Sagara, Yasuaki; Onoyama, Ichiro; Ueo, Hiroaki; Ohno, Shinji; Mori, Masaki; Mimori, Koshi; Nakayama, Keiichi I.

2015-01-01

The gene encoding F-box protein FBXW7 is frequently mutated in many human cancers. Although most previous studies have focused on the tumor-suppressive capacity of FBXW7 in tumor cells themselves, we determined that FBXW7 in the host microenvironment also suppresses cancer metastasis. Deletion of Fbxw7 in murine BM-derived stromal cells induced accumulation of NOTCH and consequent transcriptional activation of Ccl2. FBXW7-deficient mice exhibited increased serum levels of the chemokine CCL2, which resulted in the recruitment of both monocytic myeloid-derived suppressor cells and macrophages, thereby promoting metastatic tumor growth. Administration of a CCL2 receptor antagonist blocked the enhancement of metastasis in FBXW7-deficient mice. Furthermore, in human breast cancer patients, FBXW7 expression in peripheral blood was associated with serum CCL2 concentration and disease prognosis. Together, these results suggest that FBXW7 antagonizes cancer development in not only a cell-autonomous manner, but also a non-cell-autonomous manner, and that modulation of the FBXW7/NOTCH/CCL2 axis may provide a potential approach to suppression of cancer metastasis. PMID:25555218

Preclinical evaluation of WYE-687, a mTOR kinase inhibitor, as a potential anti-acute myeloid leukemia agent

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheng, Feng; Wang, Lingling; Shen, Yunfeng

Mammalian target of rapamycin (mTOR) as a potential drug target for treatment of acute myeloid leukemia (AML). Here, we investigated the potential anti-leukemic activity by WYE-687, a potent mTOR kinase inhibitor. We demonstrated that WYE-687 potently inhibited survival and proliferation of established (HL-60, U937, AML-193 and THP-1 lines) and human AML progenitor cells. Yet, same WYE-687 treatment was non-cytotoxic to the primary peripheral blood mononuclear leukocytes (PBMCs) isolated from healthy donors. WYE-687 induced caspase-dependent apoptotic death in above AML cells/progenitor cells. On the other hand, the pan-caspase inhibitor (Z-VAD-FMK), the caspase-3 specific inhibitor (Z-DEVD-FMK) or the caspase-9 specific inhibitor (z-LEHD-fmk)more » attenuated WYE-687-induced cytotoxicity. At the molecular level, WYE-687 concurrently inhibited activation of mTORC1 (p70S6K1 and S6 phosphorylations) and mTORC2 (AKT Ser-473 and FoxO1/3a phosphorylations), whiling downregulating mTORC1/2-regulated genes (Bcl-xL and hypoxia-inducible factor 1/2α) in both HL-60/U937 cells and human AML progenitor cells. In vivo, oral administration of WYE-687 potently inhibited U937 leukemic xenograft tumor growth in severe combined immunodeficient (SCID) mice, without causing significant toxicities. In summary, our results demonstrate that targeting mTORC1/2 by WYE-687 leads to potent antitumor activity in preclinical models of AML. - Highlights: • WYE-687 inhibits survival and proliferation of human AML cells/progenitor cells. • WYE-687 induces apoptotic death of human AML cells/progenitor cells. • WYE-687 inhibits mTORC1/2 activation in human AML cells/progenitor cells. • WYE-687 inhibits U937 xenograft growth in SCID mice.« less

Metronidazole reduces the expression of cytochrome P450 enzymes in HepaRG cells and cryopreserved human hepatocytes.

PubMed

Kudo, Toshiyuki; Endo, Yumiko; Taguchi, Rina; Yatsu, Masami; Ito, Kiyomi

2015-05-01

1. Blood levels of S-warfarin have been reported to be increased by concomitant administration of metronidazole (MTZ), an antiprotozoal imidazole derivative. 2. To elucidate the mechanism of this interaction and to identify other possible drug-drug interactions, we conducted an in vitro study with the human hepatoma HepaRG cells and cryopreserved human hepatocytes on the ability of MTZ to reduce the expression of cytochrome P450 (CYP) as well as nuclear receptors that regulate the expression of these enzymes. 3. HepaRG cells and cryopreserved human hepatocytes were treated with MTZ (20 to 500 µM) and were then analyzed by real-time RT-PCR to determine mRNA levels of drug-metabolizing enzymes and nuclear receptors. 4. In both cells, the expressions of CYP2C8, CYP2C9, CYP3A4 and constitutive androstane receptor (CAR) were decreased by MTZ treatment. Particularly, in HepaRG cells, their mRNA levels were decreased by MTZ treatment in a concentration-dependent manner. 5. Our findings suggest that the interaction between MTZ and S-warfarin may be due to the MTZ-induced down-regulation of CYP2C9, the primary enzyme responsible for S-warfarin hydroxylation, and CAR, which regulates CYP2C9 expression. We also found that MTZ use may alter the disposition of drugs metabolized by the CYP isozymes investigated.

HMGB1-mediated autophagy decreases sensitivity to oxymatrine in SW982 human synovial sarcoma cells

PubMed Central

Cai, Yongsong; Xu, Peng; Yang, Le; Xu, Ke; Zhu, Jialin; Wu, Xiaoqing; Jiang, Congshan; Yuan, Qiling; Wang, Bo; Li, Yuanbo; Qiu, Yusheng

2016-01-01

Oxymatrine (OMT) is a type of alkaloid extracted from a traditional Chinese medicinal herb, Sophora flavescens. Although the antitumor activities of OMT have been observed in various cancers, there are no reports regarding the effects of OMT on human synovial sarcoma. In the present study, we analyzed the antitumor activities of OMT in SW982 human synovial sarcoma cells and determine whether high mobility group box protein 1 (HMGB1)-mediated autophagy was associated with its therapeutic effects. We found that OMT exhibited antitumor activity in SW982 cells and facilitated increases in autophagy. Inhibition of autophagy by 3-MA or ATG7 siRNA increased the level of apoptosis, which indicated that OMT-induced autophagy protected cells from the cytotoxicity of OMT. Administration of OMT to SW982 cells increased the expression of HMGB1. When HMGB1 was inhibited via HMGB1-siRNA, OMT-induced autophagy was decreased, and apoptosis was increased. Furthermore, we found that HMGB1-siRNA significantly increased the expression of p-Akt and p-mTOR. OMT-induced autophagy may be mediated by the Akt/mTOR pathway, and HMGB1 plays a vital role in the regulation of autophagy. Therefore, we believe that combining OMT with an inhibitor of autophagy or HMGB1 may make OMT more effective in the treatment of human synovial sarcoma. PMID:27897164

Inhibition of monomethylarsonous acid (MMA(III))-induced cell malignant transformation through restoring dysregulated histone acetylation.

PubMed

Ge, Yichen; Gong, Zhihong; Olson, James R; Xu, Peilin; Buck, Michael J; Ren, Xuefeng

2013-10-04

Inorganic arsenic (iAs) and its high toxic metabolite, monomethylarsonous acid (MMA(III)), are able to induce malignant transformation of human cells. Chronic exposure to these chemicals is associated with an increased risk of developing multiple cancers in human. However, the mechanisms contributing to iAs/MMA(III)-induced cell malignant transformation and carcinogenesis are not fully elucidated. We recently showed that iAs/MMA(III) exposure to human cells led to a decreased level of histone acetylation globally, which was associated with an increased sensitivity to arsenic cytotoxicity. In the current study, it demonstrated that prolonged exposure to low-level MMA(III) in human urothelial cells significantly increased the expression and activity of histone deacetylases (HDACs) with an associated reduction of histone acetylation levels both globally and lysine specifically. Administration of the HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), at 4 weeks after the initial MMA(III) treatment inhibited the MMA(III)-mediated up-regulation of the expression and activities of HDACs, leading to increase histone acetylation and prevention of MMA(III)-induced malignant transformation. These new findings suggest that histone acetylation dysregulation may be a key mechanism in MMA(III)-induced malignant transformation and carcinogenesis, and that HDAC inhibitors could be targeted to prevent or treat iAs-related cancers. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Arenavirus reverse genetics for vaccine development

PubMed Central

Ortiz-Riaño, Emilio; Cheng, Benson Yee Hin; Carlos de la Torre, Juan

2013-01-01

Arenaviruses are important human pathogens with no Food and Drug Administration (FDA)-licensed vaccines available and current antiviral therapy being limited to an off-label use of the nucleoside analogue ribavirin of limited prophylactic efficacy. The development of reverse genetics systems represented a major breakthrough in arenavirus research. However, rescue of recombinant arenaviruses using current reverse genetics systems has been restricted to rodent cells. In this study, we describe the rescue of recombinant arenaviruses from human 293T cells and Vero cells, an FDA-approved line for vaccine development. We also describe the generation of novel vectors that mediate synthesis of both negative-sense genome RNA and positive-sense mRNA species of lymphocytic choriomeningitis virus (LCMV) directed by the human RNA polymerases I and II, respectively, within the same plasmid. This approach reduces by half the number of vectors required for arenavirus rescue, which could facilitate virus rescue in cell lines approved for human vaccine production but that cannot be transfected at high efficiencies. We have shown the feasibility of this approach by rescuing both the Old World prototypic arenavirus LCMV and the live-attenuated vaccine Candid#1 strain of the New World arenavirus Junín. Moreover, we show the feasibility of using these novel strategies for efficient rescue of recombinant tri-segmented both LCMV and Candid#1. PMID:23364194

Effects of oral anorexiant sibutramine on the expression of cytochromes P450s in human hepatocytes and cancer cell lines.

PubMed

Vrzal, Radim; Knoppová, Barbora; Bachleda, Petr; Dvořák, Zdeněk

2013-12-01

Sibutramine is a serotonin-norepinephrine reuptake inhibitor that was used for weight-loss management in obese patients. Even though it was officially withdrawn from the market in 2010, it is still present in some tainted weight-loss pills (as reported by US Food and Drug Administration). Thus, it is still reasonable to study the effects of this compound. The aim of this work was to investigate the potential of sibutramine to induce CYP1A1/CY3A4 in human cancer cell lines and CYP1A1/2, CYP2A6, CYP2B6, and CYP3A4 in human hepatocytes, a competent model of metabolically active cells. The levels of mRNA and protein of CYP1A1/1A2/3A4/2A6/2B6 were compared with the typical inducers, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and rifampicin (RIF) for CYP1A1/2 and for other CYPs, respectively. The mRNA and protein levels of all genes in either cancer cell lines or human hepatocytes were induced when treated with typical inducers but not with sibutramine. © 2013 Wiley Periodicals, Inc.

Human alpha-lactalbumin made lethal to tumor cells (HAMLET) kills human glioblastoma cells in brain xenografts by an apoptosis-like mechanism and prolongs survival.

PubMed

Fischer, Walter; Gustafsson, Lotta; Mossberg, Ann-Kristin; Gronli, Janne; Mork, Sverre; Bjerkvig, Rolf; Svanborg, Catharina

2004-03-15

Malignant brain tumors present a major therapeutic challenge because no selective or efficient treatment is available. Here, we demonstrate that intratumoral administration of human alpha-lactalbumin made lethal to tumor cells (HAMLET) prolongs survival in a human glioblastoma (GBM) xenograft model, by selective induction of tumor cell apoptosis. HAMLET is a protein-lipid complex that is formed from alpha-lactalbumin when the protein changes its tertiary conformation and binds oleic acid as a cofactor. HAMLET induces apoptosis in a wide range of tumor cells in vitro, but the therapeutic effect in vivo has not been examined. In this study, invasively growing human GBM tumors were established in nude rats (Han:rnu/rnu Rowett, n = 20) by transplantation of human GBM biopsy spheroids. After 7 days, HAMLET was administered by intracerebral convection-enhanced delivery for 24 h into the tumor area; and alpha-lactalbumin, the native, folded variant of the same protein, was used as a control. HAMLET reduced the intracranial tumor volume and delayed the onset of pressure symptoms in the tumor-bearing rats. After 8 weeks, all alpha-lactalbumin-treated rats had developed pressure symptoms, but the HAMLET-treated rats remained asymptomatic. Magnetic resonance imaging scans revealed large differences in tumor volume (456 versus 63 mm(3)). HAMLET caused apoptosis in vivo in the tumor but not in adjacent intact brain tissue or in nontransformed human astrocytes, and no toxic side effects were observed. The results identify HAMLET as a new candidate in cancer therapy and suggest that HAMLET should be additionally explored as a novel approach to controlling GBM progression.

A randomized phase II trial comparing two different sequence combinations of autologous vaccine and human recombinant interferon gamma and human recombinant interferon alpha2B therapy in patients with metastatic renal cell carcinoma: clinical outcome and analysis of immunological parameters.

PubMed

Schwaab, T; Heaney, J A; Schned, A R; Harris, R D; Cole, B F; Noelle, R J; Phillips, D M; Stempkowski, L; Ernstoff, M S

2000-04-01

The clinical observation of spontaneous regression in patients with renal cell carcinoma (RCC) and the response to various immunotherapeutic therapies strongly suggest a role for the host immune system in this disease. Prior studies showed that sequential administration of interferon (IFN) gamma and IFN alpha to RCC patients was safe. Clinical responses as well as immune changes in the peripheral blood mononuclear cell compartment were observed. Autologous tumor cell vaccines (AV) have also demonstrated activity in renal cell carcinoma. We hypothesize that the addition of AV to sequential IFN gamma and a therapy might improve the tumor-specific immune response by providing an appropriate source of antigen in the appropriate cytokine environment. To our knowledge, this is the first trial using AV combined with IFN alpha and IFN gamma. The purpose of this study was to evaluate the feasibility of manufacturing and administering (AV) from resected tumor samples, and administration of AV with combination IFN gamma and IFN alpha therapy. Finally, the impact on immunological parameters of these treatment options was assessed. Patients with metastatic RCC were randomly assigned to receive AV plus bCG along with a sequential administration of IFN gamma and a either together or after initiation of vaccine. Toxicity and clinical responses were evaluated. Modulations of the immune system were investigated by analyzing phenotype, cytokine mRNA expression, T cell proliferation and cytotoxicity in the peripheral blood mononuclear cell compartment. Fourteen patients with metastatic renal cell carcinoma were enrolled in this study; 9 were available for response evaluation. In a 70 day period, 3 (33%) showed mixed responses, 5 (56%) stable disease and 1 (11%) progression of disease. Toxicities were consistent with previous clinical reports. In the flow-cytometry phenotype analysis, stimulation of distinct subsets of circulating T-lymphocytes and a decrease of CD8+ T cell subsets was demonstrated. T-cell proliferation to allogeneic tumor cell stimulation improved following treatment. IL-4 and IL-5 mRNA levels were reduced in all patients after treatment. Patients who responded to treatment did not produce any IL-4 mRNA at all, before or after treatment. AV with IFNgamma and IFNalpha therapy might induce a MHC class-mediated cytotoxic T lymphocyte (CTL) response. We suggest that adequate therapy might direct T cell response toward a Th1 type response. We hypothesize a state of improved immune readiness in patients who might eventually respond to immunotherapy.

In vivo immunomodulatory, cumulative skin irritation, sensitization and effect of d-limonene on permeation of 6-mercaptopurine through transdermal drug delivery.

PubMed

Chandrashekar, N S; Hiremath, Shobha Rani Rajeev

2008-04-01

Using skin as a port for systemic drug administration, transdermal drug delivery has expanded greatly over the last two decades. Our aim was to formulate the single layer drug-in-adhesive transdermal patch for 6-mercaptopurine (6-MP). In vitro permeation study was carried out using modified Franz diffusion cell with and without of different concentration of d-limonene in human cadaver skin. In vivo immunomodulatory was carried out in mice, cumulative skin irritation, sensitization and patch adherence study was done in both mice and human subjects. 6-MP flux increased from 43+/-12.2 microg/cm2h (control) to 162.8+/-32.2 microg/cm2h (6% w/v d-limonene) data was significant (p<0.05), with decrease in the lag time to 35+/-9.3 min compared to control of 90 +/-15.3 min. In vivo immunomodulatory effect was shown in the Balb/c mice with 100 mumol/kg/body wt of animal for 5d (one dose/d) of d-limonene. WBC count of 13469 cells/mm peak was observed on 12th day, bone marrow cells of 26.3 x 10(6) cells/femur and alpha-esterase positive cells of 1259+/-328.4 cells/4000 bone marrow cells. Cumulative skin irritation, sensitisation and patch adherence in animals and human subjects showed no skin irritation and sensitization. Patch adhesion was greater than 90.0% respectively in both human subjects and mice. The percentage of human subjects with adhesive residue was significantly less with scores of zero. d-Limonene proved as good chemical enhancer by increasing in the skin permeability with shortened the lag time. It proved that therapeutic amount of 6-MP can be delivered through transdermal drug delivery.

Intracerebral adult stem cells transplantation increases brain-derived neurotrophic factor levels and protects against phencyclidine-induced social deficit in mice

PubMed Central

Barzilay, R; Ben-Zur, T; Sadan, O; Bren, Z; Taler, M; Lev, N; Tarasenko, I; Uzan, R; Gil-Ad, I; Melamed, E; Weizman, A; Offen, D

2011-01-01

Stem cell-based regenerative therapy is considered a promising cellular therapeutic approach for the patients with incurable brain diseases. Mesenchymal stem cells (MSCs) represent an attractive cell source for regenerative medicine strategies for the treatment of the diseased brain. Previous studies have shown that these cells improve behavioral deficits in animal models of neurological disorders such as Parkinson's and Huntington's diseases. In the current study, we examined the capability of intracerebral human MSCs transplantation (medial pre-frontal cortex) to prevent the social impairment displayed by mice after withdrawal from daily phencyclidine (PCP) administration (10 mg kg−1 daily for 14 days). Our results show that MSCs transplantation significantly prevented the PCP-induced social deficit, as assessed by the social preference test. In contrast, the PCP-induced social impairment was not modified by daily clozapine treatment. Tissue analysis revealed that the human MSCs survived in the mouse brain throughout the course of the experiment (23 days). Significantly increased cortical brain-derived neurotrophic factor levels were observed in the MSCs-treated group as compared with sham-operated controls. Furthermore, western blot analysis revealed that the ratio of phosphorylated Akt to Akt was significantly elevated in the MSCs-treated mice compared with the sham controls. Our results demonstrate that intracerebral transplantation of MSCs is beneficial in attenuating the social deficits induced by sub-chronic PCP administration. We suggest a novel therapeutic approach for the treatment of schizophrenia-like negative symptoms in animal models of the disorder. PMID:22832353

Pharmacokinetics and pharmacodynamics of CD4-anchoring bi-functional fusion inhibitor in monkeys.

PubMed

Liu, Xingrong; Ou, Ying C; Zhang, Jun; Ahene, Ago; Clark, Douglas; Hsieh, Su-Chun; Cooper, Matthew; Ji, Changhua

2014-03-01

This study was to characterize the pharmacokinetics (PK) and pharmacodynamics (PD) of a chimeric protein, CD4-anchoring bi-functional fusion inhibitor (CD4-BFFI), in monkeys and assess the feasibility for HIV-1 treatment in humans. The serum concentrations of CD4-BFFI and CD4 receptors were determined and modeled using a target-mediated drug disposition (TMDD) model following intravenous administration of 1 or 10 mg/kg in monkeys. In vitro CD4 internalization was examined in human peripheral blood mononuclear cells. Noncompartmental analysis showed a decrease in clearance (1.35 to 0.563 mL/h/kg) and an increase in half-lives (35 to 50 h) with increasing doses. Dose-dependent CD4 occupancy was observed. The TMDD model reasonably captured the PK/PD profiles and suggested greater degradation rate constant for the free CD4 than the bound CD4. In vitro assay showed CD4-BFFI did not reduce the internalization of cell surface CD4. The simulated serum concentrations of CD4-BFFI were 20-fold above its in vitro IC50 for HIV-1 at 3 mg/kg weekly or biweekly following subcutaneous administration in humans. The TMDD modeling and in vitro CD4 internalization study indicate that CD4-BFFI does not induce CD4 internalization and CD4-BFFI short half-life is likely due to normal CD4 internalization. The simulated human PK supports CD4-BFFI as a promising anti-HIV-1 agent.

Assessment of oral transmission using cell-free human immunodeficiency virus-1 in mice reconstituted with human peripheral blood leucocyte

PubMed Central

Nakao, Ryoma; Hanada, Nobuhiro; Asano, Toshihiko; Hara, Takashi; Abdus Salam, MD; Matin, Khairul; Shimazu, Yoshihito; Nakasone, Tadashi; Horibata, Shigeo; Aoba, Takaaki; Honda, Mitsuo; Amagasa, Teruo; Senpuku, Hidenobu

2003-01-01

Oral–genital contact is one of the risk factors for the transmission of human immunodeficiency virus (HIV) in adults. In recent reports, oral exposure to simian immunodeficiency virus (SIV) was found to have important implications for the achievement of mucosal transmission of HIV in a rhesus monkey animal model. In the present study, we aimed first to establish a small animal model which did not develop tonsils suitable for HIV oral mucosa transmission, using non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice and NOD/SCID B2mnull mice grafted with human peripheral blood leucocytes (hu-PBL) and stimulated with interleukin (IL)-4, and second to investigate whether oral exposure to cell-free R5 and X4 HIV-1 could lead to oral transmission of HIV through intact or traumatized mucosal tissues in humanized mice. Both low and high concentrations of cell-free R5 and X4 viruses failed to cause oral transmission with or without trauma in hu-PBL-NOD/SCID and NOD/SCID Β2mnull mice, which presented a number of CD4+ cells in gingival tissues and oral cavities with or without tissue injury. The present results show that IL-4-administrated NOD/SCID B2mnull mice are useful as a small-humanized model for the study of HIV oral infection, which may help to define the window of opportunity for oral transmission by the HIV virus in animal model experiments. PMID:12757623

Annexin-directed β-glucuronidase for the targeted treatment of solid tumors.

PubMed

Guillen, Katrin P; Ruben, Eliza A; Virani, Needa; Harrison, Roger G

2017-02-01

Enzyme prodrug therapy has the potential to remedy the lack of selectivity associated with the systemic administration of chemotherapy. However, most current systems are immunogenic and constrained to a monotherapeutic approach. We developed a new class of fusion proteins centered about the human enzyme β-glucuronidase (βG), capable of converting several innocuous prodrugs into chemotherapeutics. We targeted βG to phosphatidylserine on tumor cells, tumor vasculature and metastases via annexin A1/A5. Phosphatidylserine shows promise as a universal marker for solid tumors and allows for tumor type-independent targeting. To create fusion proteins, human annexin A1/A5 was genetically fused to the activity-enhancing 16a3 mutant of human βG, expressed in chemically defined, fed-batch suspension culture, and chromatographically purified. All fusion constructs achieved >95% purity with yields up to 740 μg/l. Fusion proteins displayed cancer selective cell-surface binding with cell line-dependent binding stability. One fusion protein in combination with the prodrug SN-38 glucuronide was as effective as the drug SN-38 on Panc-1 pancreatic cancer cells and HAAE-1 endothelial cells, and demonstrated efficacy against MCF-7 breast cancer cells. βG fusion proteins effectively enable localized combination therapy that can be tailored to each patient via prodrug selection, with promising clinical potential based on their near fully human design. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Aromatase inhibitors in human lung cancer therapy.

PubMed

Weinberg, Olga K; Marquez-Garban, Diana C; Fishbein, Michael C; Goodglick, Lee; Garban, Hermes J; Dubinett, Steven M; Pietras, Richard J

2005-12-15

Lung cancer is the most common cancer in the world. It is a highly lethal disease in women and men, and new treatments are urgently needed. Previous studies implicated a role of estrogens and estrogen receptors in lung cancer progression, and this steroidal growth-stimulatory pathway may be promoted by tumor expression and activity of aromatase, an estrogen synthase. We found expression of aromatase transcripts and protein in human non-small cell lung cancer (NSCLC) cells using reverse transcription-PCR and Western immunoblots, respectively. Aromatase staining by immunohistochemistry was detected in 86% of archival NSCLC tumor specimens from the clinic. Further, biological activity of aromatase was determined in NSCLC tumors using radiolabeled substrate assays as well as measure of estradiol product using ELISA. Significant activity of aromatase occurred in human NSCLC tumors, with enhanced levels in tumor cells compared with that in nearby normal cells. Lung tumor aromatase activity was inhibited by anastrozole, an aromatase inhibitor, and treatment of tumor cells in vitro with anastrozole led to significant suppression of tumor cell growth. Similarly, among ovariectomized nude mice with A549 lung tumor xenografts, administration of anastrozole by p.o. gavage for 21 days elicited pronounced inhibition of tumor growth in vivo. These findings show that aromatase is present and biologically active in human NSCLCs and that tumor growth can be down-regulated by specific inhibition of aromatase. This work may lead to development of new treatment options for patients afflicted with NSCLC.

Novel CAD/CAM rapid prototyping of next-generation biomedical devices

NASA Astrophysics Data System (ADS)

Doraiswamy, Anand

An aging population with growing healthcare needs demands multifaceted tools for diagnosis and treatment of health conditions. In the near-future, drug-administration devices, implantable devices/sensors, enhanced prosthesis, artificial and unique functional tissue constructs will become increasingly significant. Conventional technologies for mass-produced implants do not adequately take individual patient anatomy into consideration. Development of novel CAD/CAM rapid prototyping techniques may significantly accelerate progress of these devices for next-generation patient-care. In this dissertation, several novel rapid prototyping techniques have been introduced for next-generation biomedical applications. Two-photon polymerization was developed to microfabricate scaffolds for tissue engineering, microneedles for drug-delivery and ossicular replacement prostheses. Various photoplymers were evaluated for feasibility, mechanical properties, cytotoxicity, and surface properties. Laser direct write using MDW was utilized for developing microstructures of bioceramics such as hydroxyapatite, and viable mammalian osteosarcoma cells. CAD/CAM laser micromachining (CLM) was developed to engineer biointerfaces as surface recognition regions for differential adherence of cells and growth into tissue-like networks. CLM was also developed for engineering multi-cellular vascular networks. Cytotoxic evaluations and growth studies demonstrated VEGF-induced proliferation of HAAE-1 human aortic endothelial cells with inhibition of HA-VSMC human aortic smooth muscle cells. Finally, piiezoelectric inkjet printing was developed for controlled administration of natural and synthetic adhesives to overcome several problems associated with conventional tissue bonding materials, and greatly improve wound-repair in next generation eye repair, fracture fixation, organ fixation, wound closure, tissue engineering, and drug delivery devices.

Effects of Freeze-Thawing and Intravenous Infusion on Mesenchymal Stromal Cell Gene Expression.

PubMed

Hoogduijn, Martin J; de Witte, Samantha F H; Luk, Franka; van den Hout-van Vroonhoven, Mirjam C G N; Ignatowicz, Lech; Catar, Rusan; Strini, Tanja; Korevaar, Sander S; van IJcken, Wilfred F J; Betjes, Michiel G H; Franquesa, Marcella; Moll, Guido; Baan, Carla C

2016-04-15

Mesenchymal stromal cells (MSC) are increasingly used as an investigative therapeutic product for immune disorders and degenerative disease. Typically, MSC are isolated from human tissue, expanded in culture, and cryopreserved until usage. The safety and efficacy of MSC therapy will depend on the phenotypical and functional characteristics of MSC. The freeze-thawing procedure may change these characteristics. Furthermore, the cells encounter a microenvironment after administration that may impact their properties. It has been demonstrated that the majority of MSC localize to the lungs after intravenous infusion, making this the site to study the effects of the in vivo milieu on administered MSC. In this study, we investigated the effect of freeze-thawing and the mouse lung microenvironment on human adipose tissue-derived MSC. There were effects of freeze-thawing on the whole genome expression profile of MSC, although the effects did not exceed interdonor differences. There were no major changes in the expression of hemostatic regulators on transcriptional level, but significantly increased expression of procoagulant tissue factor on the surface of thawed adipose MSC, correlating with increased procoagulant activity of thawed cells. Exposure for 2 h to the lung microenvironment had a major effect on MSC gene expression and affected several immunological pathways. This indicates that MSC undergo functional changes shortly after infusion and this may influence the efficacy of MSC to modulate inflammatory responses. The results of this study demonstrate that MSC rapidly alter in response to the local milieu and disease-specific conditions may shape MSC after administration.

Neuroglobin overexpression plays a pivotal role in neuroprotection through mitochondrial raft-like microdomains in neuroblastoma SK-N-BE2 cells.

PubMed

Garofalo, Tina; Ferri, Alberto; Sorice, Maurizio; Azmoon, Pardis; Grasso, Maria; Mattei, Vincenzo; Capozzi, Antonella; Manganelli, Valeria; Misasi, Roberta

2018-04-01

Since stressing conditions induce a relocalization of endogenous human neuroglobin (NGB) to mitochondria, this research is aimed to evaluate the protective role of NGB overexpression against neurotoxic stimuli, through mitochondrial lipid raft-associated complexes. To this purpose, we built a neuronal model of oxidative stress by the use of human dopaminergic neuroblastoma cells, SK-N-BE2, stably overexpressing NGB by transfection and treated with 1-methyl-4-phenylpyridinium ion (MPP+). We preliminary observed the redistribution of NGB to mitochondria following MPP+ treatment. The analysis of mitochondrial raft-like microdomains revealed that, following MPP+ treatment, NGB translocated to raft fractions (Triton X-100-insoluble), where it interacts with ganglioside GD3. Interestingly, the administration of agents capable of perturbating microdomain before MPP+ treatment, significantly affected viability in SK-N-BE2-NGB cells. The overexpression of NGB was able to abrogate the mitochondrial injuries on complex IV activity or mitochondrial morphology induced by MPP+ administration. The protective action of NGB on mitochondria only takes place if the mitochondrial lipid(s) rafts-like microdomains are intact, indeed NGB fails to protect complex IV activity when purified mitochondria were treated with the lipid rafts disruptor methyl-β-cyclodextrin. Thus, our unique in vitro model of stably transfected cells overexpressing endogenous NGB allowed us to suggest that the role in neuroprotection played by NGB is reliable only through interaction with mitochondrial lipid raft-associated complexes. Copyright © 2018 Elsevier Inc. All rights reserved.

Evaluation of a Novel Renewable Hepatic Cell Model for Prediction of Clinical CYP3A4 Induction Using a Correlation-Based Relative Induction Score Approach.

PubMed

Zuo, Rongjun; Li, Feng; Parikh, Sweta; Cao, Li; Cooper, Kirsten L; Hong, Yulong; Liu, Jin; Faris, Ronald A; Li, Daochuan; Wang, Hongbing

2017-02-01

Metabolism enzyme induction-mediated drug-drug interactions need to be carefully characterized in vitro for drug candidates to predict in vivo safety risk and therapeutic efficiency. Currently, both the Food and Drug Administration and European Medicines Agency recommend using primary human hepatocytes as the gold standard in vitro test system for studying the induction potential of candidate drugs on cytochrome P450 (CYP), CYP3A4, CYP1A2, and CYP2B6. However, primary human hepatocytes are known to bear inherent limitations such as limited supply and large lot-to-lot variations, which result in an experimental burden to qualify new lots. To overcome these shortcomings, a renewable source of human hepatocytes (i.e., Corning HepatoCells) was developed from primary human hepatocytes and was evaluated for in vitro CYP3A4 induction using methods well established by the pharmaceutical industry. HepatoCells have shown mature hepatocyte-like morphology and demonstrated primary hepatocyte-like response to prototypical inducers of all three CYP enzymes with excellent consistency. Importantly, HepatoCells retain a phenobarbital-responsive nuclear translocation of human constitutive androstane receptor from the cytoplasm, characteristic to primary hepatocytes. To validate HepatoCells as a useful tool to predict potential clinical relevant CYP3A4 induction, we tested three different lots of HepatoCells with a group of clinical strong, moderate/weak CYP3A4 inducers, and noninducers. A relative induction score calibration curve-based approach was used for prediction. HepatoCells showed accurate prediction comparable to primary human hepatocytes. Together, these results demonstrate that Corning HepatoCells is a reliable in vitro model for drug-drug interaction studies during the early phase of drug testing. Copyright © 2017 by The Author(s).

Trafficking of adeno-associated virus vectors across a model of the blood-brain barrier; a comparative study of transcytosis and transduction using primary human brain endothelial cells.

PubMed

Merkel, Steven F; Andrews, Allison M; Lutton, Evan M; Mu, Dakai; Hudry, Eloise; Hyman, Bradley T; Maguire, Casey A; Ramirez, Servio H

2017-01-01

Developing therapies for central nervous system (CNS) diseases is exceedingly difficult because of the blood-brain barrier (BBB). Notably, emerging technologies may provide promising new options for the treatment of CNS disorders. Adeno-associated virus serotype 9 (AAV9) has been shown to transduce cells in the CNS following intravascular administration in rodents, cats, pigs, and non-human primates. These results suggest that AAV9 is capable of crossing the BBB. However, mechanisms that govern AAV9 transendothelial trafficking at the BBB remain unknown. Furthermore, possibilities that AAV9 may transduce brain endothelial cells or affect BBB integrity still require investigation. Using primary human brain microvascular endothelial cells as a model of the human BBB, we performed transduction and transendothelial trafficking assays comparing AAV9 to AAV2, a serotype that does not cross the BBB or transduce endothelial cells effectively in vivo. Results of our in vitro studies indicate that AAV9 penetrates brain microvascular endothelial cells barriers more effectively than AAV2, but has reduced transduction efficiency. In addition, our data suggest that (i) AAV9 penetrates endothelial barriers through an active, cell-mediated process, and (ii) AAV9 fails to disrupt indicators of BBB integrity such as transendothelial electrical resistance, tight junction protein expression/localization, and inflammatory activation status. Overall, this report shows how human brain endothelial cells configured in BBB models can be utilized for evaluating transendothelial movement and transduction kinetics of various AAV capsids. Importantly, the use of a human in vitro BBB model can provide import insight into the possible effects that candidate AVV gene therapy vectors may have on the status of BBB integrity. Read the Editorial Highlight for this article on page 192. © 2016 International Society for Neurochemistry.

Functional characterization of CFI-402257, a potent and selective Mps1/TTK kinase inhibitor, for the treatment of cancer.

PubMed

Mason, Jacqueline M; Wei, Xin; Fletcher, Graham C; Kiarash, Reza; Brokx, Richard; Hodgson, Richard; Beletskaya, Irina; Bray, Mark R; Mak, Tak W

2017-03-21

Loss of cell-cycle control is a hallmark of human cancer. Cell-cycle checkpoints are essential for maintaining genome integrity and balanced growth and division. They are specifically deregulated in cancer cells and contain regulators that represent potential therapeutic targets. Monopolar spindle 1 (Mps1; also known as TTK protein kinase) is a core component of the spindle assembly checkpoint (SAC), a genome-surveillance mechanism that is important for cell survival, and has emerged as a candidate target for anticancer therapy. Here, we report the cellular and antitumor effects of CFI-402257, a potent (Mps1 K i = 0.09 ± 0.02 nM; cellular Mps1 EC 50 = 6.5 ± 0.5 nM), highly selective, and orally active small-molecule inhibitor of Mps1 that was identified through a drug-discovery program. Human cancer cells treated with CFI-402257 exhibit effects consistent with Mps1 kinase inhibition, specifically SAC inactivation, leading to chromosome missegregation, aneuploidy, and ultimately cell death. Oral administration of CFI-402257 in monotherapy or in combination with an anti-programmed cell death 1 (PD-1) antibody in mouse models of human cancer results in inhibition of tumor growth at doses that are well-tolerated. Our findings provide a rationale for the clinical evaluation of CFI-402257 in patients with solid tumors.

Functional characterization of CFI-402257, a potent and selective Mps1/TTK kinase inhibitor, for the treatment of cancer

PubMed Central

Mason, Jacqueline M.; Wei, Xin; Fletcher, Graham C.; Kiarash, Reza; Brokx, Richard; Hodgson, Richard; Beletskaya, Irina; Bray, Mark R.; Mak, Tak W.

2017-01-01

Loss of cell-cycle control is a hallmark of human cancer. Cell-cycle checkpoints are essential for maintaining genome integrity and balanced growth and division. They are specifically deregulated in cancer cells and contain regulators that represent potential therapeutic targets. Monopolar spindle 1 (Mps1; also known as TTK protein kinase) is a core component of the spindle assembly checkpoint (SAC), a genome-surveillance mechanism that is important for cell survival, and has emerged as a candidate target for anticancer therapy. Here, we report the cellular and antitumor effects of CFI-402257, a potent (Mps1 Ki = 0.09 ± 0.02 nM; cellular Mps1 EC50 = 6.5 ± 0.5 nM), highly selective, and orally active small-molecule inhibitor of Mps1 that was identified through a drug-discovery program. Human cancer cells treated with CFI-402257 exhibit effects consistent with Mps1 kinase inhibition, specifically SAC inactivation, leading to chromosome missegregation, aneuploidy, and ultimately cell death. Oral administration of CFI-402257 in monotherapy or in combination with an anti-programmed cell death 1 (PD-1) antibody in mouse models of human cancer results in inhibition of tumor growth at doses that are well-tolerated. Our findings provide a rationale for the clinical evaluation of CFI-402257 in patients with solid tumors. PMID:28270606

Enhanced antitumor effect of YM872 and AG1296 combination treatment on human glioblastoma xenograft models.

PubMed

Watanabe, Takashi; Ohtani, Toshiyuki; Aihara, Masanori; Ishiuchi, Shogo

2013-04-01

Blockade of Ca(++)-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) inhibits the proliferation of human glioblastoma by inhibiting Akt phosphorylation, which is independent of the phosphatidylinositol 3-kinase pathway. Inhibiting platelet-derived growth factor receptor (PDGFR)-mediated phosphorylation causes growth inhibition in glioblastoma cells. The authors of this study investigated the effects of YM872 and AG1296, singly and in combination and targeting different pathways upstream of Akt, on Akt-mediated tumor growth in glioblastoma cells in vivo and in vitro. The expression of AMPAR, PDGFR, and c-kit in glioblastoma cells was analyzed via immunofluorescence. Glioblastoma cells, both in culture and in xenografts grown in mice, were treated with YM872 and AG1296, singly or in combination. Inhibition of tumor growth was observed after treatment in the xenograft model. Cell proliferation assays were performed using anti-Ki 67 antibody in vivo and in vitro. The CD34-positive tumor vessel counts within the vascular hot spots of tumor specimens were evaluated. Phosphorylation of Akt was studied using Western blot analysis. Combined administration of YM872 and AG1296 had a significant enhanced effect on the inhibition of cell proliferation and reduction of tumor vascularity in the xenograft model. These agents singly and in combination demonstrated a significant reduction of Akt phosphorylation at Ser473 and inhibition of tumor proliferation in vitro, although combined administration had no enhanced antitumor effects. The strongly enhanced antitumor effect of this combination therapy in vivo rather than in vitro may be attributable to disruption of the aberrant vascular niche. This combination therapy might provide substantial benefits to patients with glioblastoma.

2005 Donor Eligibility Requirements: Unintended Consequences for Stem Cell Development.

PubMed

Couture, Larry A; Carpenter, Melissa K

2015-10-01

Several human embryonic stem cell (hESC)-derived cell therapeutics have entered clinical testing and more are in various stages of preclinical development. The U.S. Food and Drug Administration (FDA) regulates these products under existing regulations and has stated that these products do not constitute a new class of biologic. However, as human tissue, hESCs are subject to regulations that were developed before hESCs were first described. The regulations have not been revised since 2005, well before the first hESC-derived product entered clinical studies. The current regulations require donors of hESCs to be tested in the same manner as donors of tissues intended for transplantation. However, because hESC-derived cell products are more than minimally manipulated, they are also subject to the same end-of-production release testing as most other biologic agents. In effect, this makes hESC products subject to redundant testing. No other biologic is subject to a similar testing requirement. Furthermore, the regulations that require donor testing are specifically applicable to hESC cells harvested from donors after a date in 2005. It is unclear which regulations cover hESCs harvested before 2005. Ambiguity in the guidelines and redundant testing requirements have unintentionally created a burdensome regulatory paradigm for these products and reluctance on the part of developers to invest in these promising therapeutics. We propose a simple solution that would address FDA safety concerns, eliminate regulatory uncertainty and risk, and provide flexibility for the FDA in the regulation of hESC-derived cell therapies. Regulatory ambiguity concerning donor eligibility screening and testing requirements for human embryonic stem cell lines, in particular those lines created before 2005, are causing significant concern for drug developers. Technically, most of these lines fail to meet eligibility under U.S. Food and Drug Administration (FDA) rules for product licensure, and many developers are unaware that FDA approval to begin trials under an exemption is not an assurance that the FDA will grant licensure of the product. This Perspective outlines the ambiguity and the problem it has caused and proposes a workable solution. The intent is to generate stakeholder and FDA discussion on this issue. ©AlphaMed Press.

CAR-Engineered NK Cells Targeting Wild-Type EGFR and EGFRvIII Enhance Killing of Glioblastoma and Patient-Derived Glioblastoma Stem Cells.

PubMed

Han, Jianfeng; Chu, Jianhong; Keung Chan, Wing; Zhang, Jianying; Wang, Youwei; Cohen, Justus B; Victor, Aaron; Meisen, Walter H; Kim, Sung-hak; Grandi, Paola; Wang, Qi-En; He, Xiaoming; Nakano, Ichiro; Chiocca, E Antonio; Glorioso, Joseph C; Kaur, Balveen; Caligiuri, Michael A; Yu, Jianhua

2015-07-09

Glioblastoma (GB) remains the most aggressive primary brain malignancy. Adoptive transfer of chimeric antigen receptor (CAR)-modified immune cells has emerged as a promising anti-cancer approach, yet the potential utility of CAR-engineered natural killer (NK) cells to treat GB has not been explored. Tumors from approximately 50% of GB patients express wild-type EGFR (wtEGFR) and in fewer cases express both wtEGFR and the mutant form EGFRvIII; however, previously reported CAR T cell studies only focus on targeting EGFRvIII. Here we explore whether both wtEGFR and EGFRvIII can be effectively targeted by CAR-redirected NK cells to treat GB. We transduced human NK cell lines NK-92 and NKL, and primary NK cells with a lentiviral construct harboring a second generation CAR targeting both wtEGFR and EGFRvIII and evaluated the anti-GB efficacy of EGFR-CAR-modified NK cells. EGFR-CAR-engineered NK cells displayed enhanced cytolytic capability and IFN-γ production when co-cultured with GB cells or patient-derived GB stem cells in an EGFR-dependent manner. In two orthotopic GB xenograft mouse models, intracranial administration of NK-92-EGFR-CAR cells resulted in efficient suppression of tumor growth and significantly prolonged the tumor-bearing mice survival. These findings support intracranial administration of NK-92-EGFR-CAR cells represents a promising clinical strategy to treat GB.

Only Follow-Up of Memory B Cells Helps Monitor Rituximab Administration to Patients with Neuromyelitis Optica Spectrum Disorders.

PubMed

Lebrun, Christine; Cohen, Mikael; Rosenthal-Allieri, Maria Alessandra; Bresch, Saskia; Benzaken, Sylvia; Marignier, Romain; Seitz-Polski, Barbara; Ticchioni, Michel

2018-06-07

Neuromyelitis optica spectrum disorders (NMOSD) are identified as a spectrum of inflammatory demyelinating disorders involving the brain, spinal cord and optic nerves. These disorders require early diagnosis and highly active immunosuppressive treatment. Rituximab (RTX) has demonstrated efficacy in limiting relapse in NMOSD when using several administration schedules. We questioned if the CD19+ CD27+ memory B cell count was a more reliable marker to monitor RTX administration than the RTX plasma level and CD19+ B cell count. We analyzed 125 blood samples from 17 NMOSD patients treated with RTX and also measured the level of anti-aquaporine-4 antibodies (anti-AQP-4 Abs), human anti-chimeric antibodies to the murine fragment of RTX (HACA-RTX Abs), and the RTX concentration. The mean follow-up time of the cohort was 7.4 (2-16) years. All patients improved with a mean EDSS going from 4 (1-8.5) to 2.7 (1-5.5). The mean interval between RTX infusions was 9.6 months with identification of prolonged responders. Total CD19+ B cell detection with the routine technique did not correlate to re-emergence of CD19+ CD27+ memory B cells. The RTX residual concentration did not correlate with the CD19+ CD27+ memory B cell count or with anti-RTX antibody production. In contrast to total CD19+ cell, detected with the routine technique, CD19+ CD27+ memory B cells are a reliable marker for biological relapse and allow a decrease in the frequency of infusions.

Oral administration of an HSP90 inhibitor, 17-DMAG, intervenes tumor-cell infiltration into multiple organs and improves survival period for ATL model mice

PubMed Central

Ikebe, E; Kawaguchi, A; Tezuka, K; Taguchi, S; Hirose, S; Matsumoto, T; Mitsui, T; Senba, K; Nishizono, A; Hori, M; Hasegawa, H; Yamada, Y; Ueno, T; Tanaka, Y; Sawa, H; Hall, W; Minami, Y; Jeang, K T; Ogata, M; Morishita, K; Hasegawa, H; Fujisawa, J; Iha, H

2013-01-01

In the peripheral blood leukocytes (PBLs) from the carriers of the human T-lymphotropic virus type-1 (HTLV-1) or the patients with adult T-cell leukemia (ATL), nuclear factor kappaB (NF-κB)-mediated antiapoptotic signals are constitutively activated primarily by the HTLV-1-encoded oncoprotein Tax. Tax interacts with the I κB kinase regulatory subunit NEMO (NF-κB essential modulator) to activate NF-κB, and this interaction is maintained in part by a molecular chaperone, heat-shock protein 90 (HSP90), and its co-chaperone cell division cycle 37 (CDC37). The antibiotic geldanamycin (GA) inhibits HSP90's ATP binding for its proper interaction with client proteins. Administration of a novel water-soluble and less toxic GA derivative, 17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG), to Tax-expressing ATL-transformed cell lines, C8166 and MT4, induced significant degradation of Tax. 17-DMAG also facilitated growth arrest and cellular apoptosis to C8166 and MT4 and other ATL cell lines, although this treatment has no apparent effects on normal PBLs. 17-DMAG also downregulated Tax-mediated intracellular signals including the activation of NF-κB, activator protein 1 or HTLV-1 long terminal repeat in Tax-transfected HEK293 cells. Oral administration of 17-DMAG to ATL model mice xenografted with lymphomatous transgenic Lck-Tax (Lck proximal promoter-driven Tax transgene) cells or HTLV-1-producing tumor cells dramatically attenuated aggressive infiltration into multiple organs, inhibited de novo viral production and improved survival period. These observations identified 17-DMAG as a promising candidate for the prevention of ATL progression. PMID:23955587

Nanoparticles of barium induce apoptosis in human phagocytes

PubMed Central

Mores, Luana; França, Eduardo Luzia; Silva, Núbia Andrade; Suchara, Eliane Aparecida; Honorio-França, Adenilda Cristina

2015-01-01

Purpose Nutrients and immunological factors of breast milk are essential for newborn growth and the development of their immune system, but this secretion can contain organic and inorganic toxins such as barium. Colostrum contamination with barium is an important issue to investigate because this naturally occurring element is also associated with human activity and industrial pollution. The study evaluated the administration of barium nanoparticles to colostrum, assessing the viability and functional activity of colostral mononuclear phagocytes. Methods Colostrum was collected from 24 clinically healthy women (aged 18–35 years). Cell viability, superoxide release, intracellular Ca2+ release, and phagocyte apoptosis were analyzed in the samples. Results Treatment with barium lowered mononuclear phagocyte viability, increased superoxide release, and reduced intracellular calcium release. In addition, barium increased cell death by apoptosis. Conclusion These data suggest that nanoparticles of barium in colostrum are toxic to cells, showing the importance of avoiding exposure to this element. PMID:26451108

Effect of Lactobacilli on Paracellular Permeability in the Gut

PubMed Central

Ahrne, Siv; Hagslatt, Marie-Louise Johansson

2011-01-01

Paracellular permeability is determined by the complex structures of junctions that are located between the epithelial cells. Already in 1996, it was shown that the human probiotic strain Lactobacillus plantarum 299v and the rat-originating strain Lactobacillus reuteri R2LC could reduce this permeability in a methotrexate-induced colitis model in the rat. Subsequently, many animal models and cell culture systems have shown indications that lactobacilli are able to counteract increased paracellular permeability evoked by cytokines, chemicals, infections, or stress. There have been few human studies focusing on the effect of lactobacilli on intestinal paracellular permeability but recently it has been shown that they could influence the tight junctions. More precisely, short-term administration of L. plantarum WCSF1 to healthy volunteers increased the relocation of occludin and ZO-1 into the tight junction area between duodenal epithelial cells. PMID:22254077

Nanoparticles of barium induce apoptosis in human phagocytes.

PubMed

Mores, Luana; França, Eduardo Luzia; Silva, Núbia Andrade; Suchara, Eliane Aparecida; Honorio-França, Adenilda Cristina

2015-01-01

Nutrients and immunological factors of breast milk are essential for newborn growth and the development of their immune system, but this secretion can contain organic and inorganic toxins such as barium. Colostrum contamination with barium is an important issue to investigate because this naturally occurring element is also associated with human activity and industrial pollution. The study evaluated the administration of barium nanoparticles to colostrum, assessing the viability and functional activity of colostral mononuclear phagocytes. Colostrum was collected from 24 clinically healthy women (aged 18-35 years). Cell viability, superoxide release, intracellular Ca(2+) release, and phagocyte apoptosis were analyzed in the samples. Treatment with barium lowered mononuclear phagocyte viability, increased superoxide release, and reduced intracellular calcium release. In addition, barium increased cell death by apoptosis. These data suggest that nanoparticles of barium in colostrum are toxic to cells, showing the importance of avoiding exposure to this element.

Detection of PLGA-based nanoparticles at a single-cell level by synchrotron radiation FTIR spectromicroscopy and correlation with X-ray fluorescence microscopy

PubMed Central

Pascolo, Lorella; Bortot, Barbara; Benseny-Cases, Nuria; Gianoncelli, Alessandra; Tosi, Giovanni; Ruozi, Barbara; Rizzardi, Clara; De Martino, Eleonora; Vandelli, Maria Angela; Severini, Giovanni Maria

2014-01-01

Poly-lactide-co-glycolide (PLGA) is one of the few polymers approved by the US Food and Drug Administration as a carrier for drug administration in humans; therefore, it is one of the most used materials in the formulation of polymeric nanoparticles (NPs) for therapeutic purposes. Because the cellular uptake of polymeric NPs is a hot topic in the nanomedicine field, the development of techniques able to ensure incontrovertible evidence of the presence of NPs in the cells plays a key role in gaining understanding of their therapeutic potential. On the strength of this premise, this article aims to evaluate the application of synchrotron radiation-based Fourier transform infrared spectroscopy (SR-FTIR) spectromicroscopy and SR X-ray fluorescence (SR-XRF) microscopy in the study of the in vitro interaction of PLGA NPs with cells. To reach this goal, we used PLGA NPs, sized around 200 nm and loaded with superparamagnetic iron oxide NPs (PLGA-IO-NPs; Fe3O4; size, 10–15 nm). After exposing human mesothelial (MeT5A) cells to PLGA-IO-NPs (0.1 mg/mL), the cells were analyzed after fixation both by SR-FTIR spectromicroscopy and SR-XRF microscopy setups. SR-FTIR-SM enabled the detection of PLGA NPs at single-cell level, allowing polymer detection inside the biological matrix by the characteristic band in the 1,700–2,000 cm−1 region. The precise PLGA IR-signature (1,750 cm−1 centered pick) also was clearly evident within an area of high amide density. SR-XRF microscopy performed on the same cells investigated under SR-FTIR microscopy allowed us to put in evidence the Fe presence in the cells and to emphasize the intracellular localization of the PLGA-IO-NPs. These findings suggest that SR-FTIR and SR-XRF techniques could be two valuable tools to follow the PLGA NPs’ fate in in vitro studies on cell cultures. PMID:24944512

Nonclinical evaluation of the potential for mast cell activation by an erythropoietin analog

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weaver, James L., E-mail: James.Weaver@fda.hhs.gov; Boyne, Michael, E-mail: mboyne@biotechlogic.com; Pang, Eric, E-mail: Eric.Pang@fda.hhs.gov

The erythropoietin analog peginesatide was withdrawn from marketing due to unexpected severe anaphylactic reactions associated with administration of the multi-use formulation. The adverse events occurred rapidly following the first ever administration of the drug with most affected patients becoming symptomatic in less than 30 min. This is most consistent with an anaphylactoid reaction due to direct activation of mast cells. Laboratory evaluation was undertaken using rat peritoneal mast cells as the model system. Initial studies showed that high concentrations of the formulated drug as well as formulated vehicle alone could cause mast cell degranulation as measured by histamine release. Themore » purified active drug was not able to cause histamine release whereas the vehicle filtrate and lab created drug vehicle were equally potent at causing histamine release. Individual formulations of vehicle leaving one component out showed that histamine release was due to phenol. Dose response studies with phenol showed a very sharp dose response curve that was similar in three buffer systems. Cellular analysis by flow cytometry showed that the histamine release was not due to cell death, and that changes in light scatter parameters consistent with degranulation were rapidly observed. Limited testing with primary human mast cells showed a similar dose response of histamine release with exposure to phenol. To provide in vivo confirmation, rats were injected with vehicle formulated with various concentrations of phenol via a jugular vein cannula. Significant release of histamine was detected in blood samples taken 2 min after dosing at the highest concentrations tested. - Highlights: • Peginesatide caused severe anaphylactoid reactions in 0.2% of patients. • Both formulated drug and vehicle cause degranulation of rat mast cells. • Phenol was identified as the vehicle component causing degranulation. • Human mast cells show similar dose response to phenol as rat mast cells. • Histamine release could be caused in vivo in rats by rapid phenol injection.« less

Bovine lactoferricin induces caspase-independent apoptosis in human B-lymphoma cells and extends the survival of immune-deficient mice bearing B-lymphoma xenografts.

PubMed

Furlong, Suzanne J; Mader, Jamie S; Hoskin, David W

2010-06-01

Although current treatments based on the use of B-cell-specific anti-CD20 monoclonal antibodies and aggressive combinatorial chemotherapy have improved the survival of patients suffering from B-cell non-Hodgkin's lymphoma (NHL), some individuals fail to respond to treatment and relapses remain common. New and more effective treatments for B-cell NHL are therefore required. Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide that is cytotoxic for several human tumor cell lines but does not harm healthy cells. Here we show that in vitro treatment with LfcinB caused Raji and Ramos human B-lymphoma cells to die by apoptosis, as indicated by DNA fragmentation, chromatin condensation, and nuclear disintegration. LfcinB killed B-lymphoma cells more efficiently at low serum concentrations and was inhibited in the presence of exogenous bovine serum albumin, suggesting partial neutralization of cationic LfcinB by anionic serum components. LfcinB-induced apoptosis in B-lymphoma cells was caspase-independent since caspase-3 activation was not detected by Western blotting and the general caspase inhibitor z-VAD-fmk did not prevent LfcinB-induced DNA fragmentation. Importantly, immune-deficient SCID/beige mice that were inoculated intravenously with Ramos B-lymphoma cells in order to model B-cell NHL exhibited extended survival following systemic administration of LfcinB, indicating that LfcinB warrants further investigation as a novel therapeutic agent for the possible treatment of B-cell NHL. Copyright 2010 Elsevier Inc. All rights reserved.

Anti-cancer activity of bromelain nanoparticles by oral administration.

PubMed

Bhatnagar, Priyanka; Patnaik, Soma; Srivastava, Amit K; Mudiam, Mohan K R; Shukla, Yogeshwer; Panda, Amulya K; Pant, Aditya B; Kumar, Pradeep; Gupta, Kailash C

2014-12-01

Oral administration of anti-cancer drugs is an effective alternative to improve their efficacy and reduce undesired toxicity. Bromelain (BL) is known as an effective anti-cancer phyto-therapeutic agent, however, its activity is reduced upon oral administration. In addressing the issue, BL was encapsulated in Poly(lactic-co-glycolic acid) (PLGA) to formulate nanoparticles (NPs). Further, the NPs were coated with Eudragit L30D polymer to introduce stability against the gastric acidic conditions. The resultant coated NPs were characterized for BL entrapment, proteolytic activity and mean particle size. The stability and release pattern of NPs were evaluated under simulated gastrointestinal tract (GIT) pH conditions. Cytotoxicity studies carried out in human cell lines of diverse origin have shown significant dose advantage (-7-10 folds) with NPs in reducing the IC50 values compared with free BL. The cellular uptake of NPs in MCF-7, HeLa and Caco-2 cells monolayer was significantly enhanced several folds as compared to free BL. Altered expression of marker proteins associated with apoptosis and cell death (P53, P21, Bcl2, Bax) also confirmed the enhanced anti-carcinogenic potential of formulated NPs. Oral administration of NPs reduced the tumor burden of Ehrlich ascites carcinoma (EAC) in Swiss albino mice and also increased their life-span (160.0 ± 5.8%) when compared with free BL (24 ± 3.2%). The generation of reactive oxygen species, induction of apoptosis and impaired mitochondrial membrane potential in EAC cells treated with NPs confirmed the suitability of Eudragit coated BL-NPs as a promising candidate for oral chemotherapy.

Influence of hydrogen-occluding-silica on migration and apoptosis in human esophageal cells in vitro.

PubMed

Li, Qiang; Tanaka, Yoshiharu; Miwa, Nobuhiko

2017-01-01

In the last decade, many studies have shown that hydrogen gas or hydrogen water can reduce the levels of reactive oxygen species in the living body. Molecular hydrogen has antioxidant and antiapoptotic effects and a preventive effect on oxidative stress-induced cell death. In the present study, we investigated solidified hydrogen-occluding-silica (H 2 -silica) that can release molecular hydrogen into cell culture medium because the use of hydrogen gas has strict handling limitations in hospital and medical facilities and laboratories, owing to its physicochemical characteristics. Human esophageal squamous cell carcinoma (KYSE-70) cells and normal human esophageal epithelial cells (HEEpiCs) were used to investigate the effects of H 2 -silica on cell viability and proliferation. Cell migration was examined with wound healing and culture-insert migration assays. The intracellular levels of reactive oxygen species were evaluated with a nitroblue tetrazolium assay. To assess the apoptotic status of the cells, the Bax/Bcl-2 ratio and cleaved caspase-3 were analyzed by western blot. The results showed that KYSE-70 cells and HEEpiCs were generally inhibited by H 2 -silica administration, and there was a significant proliferation-inhibitory effect in an H 2 -silica concentration-dependent manner compared with the control group ( P < 0.05) in KYSE-70. Apoptosis-inducing effect on KYSE-70 cells was observed in 10, 300, 600, and 1,200 ppm H 2 -silica, and only 1,200 ppm H 2 -silica caused a 2.4-fold increase in apoptosis in HEEpiCs compared with the control group as the index of Bax/Bcl-2. H 2 silica inhibited cell migration in KYSE-70 cells, and high concentrations had a cytotoxic effect on normal cells. These findings should provide insights into the mechanism of inhibition of H 2 -silica on human cancer cells in vitro .

Analyses of functions of an anti-PD-L1/TGFβR2 bispecific fusion protein (M7824).

PubMed

Jochems, Caroline; Tritsch, Sarah R; Pellom, Samuel Troy; Su, Zhen; Soon-Shiong, Patrick; Wong, Hing C; Gulley, James L; Schlom, Jeffrey

2017-09-26

M7824 (MSB0011359C) is a novel first-in-class bifunctional fusion protein consisting of a fully human IgG1 anti-PD-L1 monoclonal antibody (with structural similarities to avelumab) linked to the extracellular domain of two TGFβ receptor 2 (TGFβR2) molecules serving as a TGFβ Trap. Avelumab has demonstrated clinical activity in a range of human cancers and has been approved by the Food and Drug Administration for the therapy of Merkel cell and bladder carcinomas. Preclinical studies have shown this anti-PD-L1 is capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC). In the studies reported here, it is shown that M7824 is also capable of mediating ADCC of a wide range of human carcinoma cells in vitro , employing natural killer (NK) cells as effectors, albeit not as potent as anti-PD-L1 employing some tumor cells as targets. The addition of the IL-15 superagonist fusion protein complex ALT-803 enhanced the ADCC capacity of both anti-PD-L1 and M7824, and to levels that both agents now demonstrated similar levels of ADCC of tumor cells. TGFβ is a known immunosuppressive entity. Studies reported here show TGFβ1 induced reduction of several NK activation markers as well as reduction of endogenous NK lytic activity and NK-mediated ADCC of tumor cells. These phenomena could be reduced or mitigated, however, by M7824, but not by anti-PD-L1. M7824, but not anti-PD-L1, was also shown to reduce the immunosuppressive activity of regulatory T cells on human CD4 + T-cell proliferation. These studies thus demonstrate the dual functionalities of M7824 and provide the rationale for its further clinical development.

Paeonia lactiflora Enhances the Adhesion of Trophoblast to the Endometrium via Induction of Leukemia Inhibitory Factor Expression

PubMed Central

Park, Mi-Ju; Lee, Kyu Sup; Yoon, Youngjin; Kim, Hyung Sik; Lee, Jun Hee; Kwon, Sang-Mo; Lee, Syng-Ook; Kim, Keuk-Jun; Baek, Jin-Ho; Ha, Ki-Tae

2016-01-01

In the present study, we investigated the role of Paeonia lactiflora Pall. extract on embryo implantation in vitro and in vivo. A polysaccharides depleted-water extract of P. lactiflora (PL-PP) increased LIF expression in human endometrial Ishikawa cells at non-cytotoxic doses. PL-PP significantly increased the adhesion of the human trophectoderm-derived JAr spheroids to endometrial Ishikawa cells. PL-PP-induced LIF expression was decreased in the presence of a p38 kinase inhibitor SB203580 and an MEK/ERK inhibitor U0126. Furthermore, endometrial LIF knockdown by shRNA reduced the expression of integrins β3 and β5 and adhesion of JAr spheroids to Ishikawa cells. In vivo administration of PL-PP restored the implantation of mouse blastocysts in a mifepristone-induced implantation failure mice model. Our results demonstrate that PL-PP increases LIF expression via the p38 and MEK/ERK pathways and favors trophoblast adhesion to endometrial cells. PMID:26839969

Improving the efficacy and safety of biologic drugs with tolerogenic nanoparticles

NASA Astrophysics Data System (ADS)

Kishimoto, Takashi K.; Ferrari, Joseph D.; Lamothe, Robert A.; Kolte, Pallavi N.; Griset, Aaron P.; O'Neil, Conlin; Chan, Victor; Browning, Erica; Chalishazar, Aditi; Kuhlman, William; Fu, Fen-Ni; Viseux, Nelly; Altreuter, David H.; Johnston, Lloyd; Maldonado, Roberto

2016-10-01

The development of antidrug antibodies (ADAs) is a common cause for the failure of biotherapeutic treatments and adverse hypersensitivity reactions. Here we demonstrate that poly(lactic-co-glycolic acid) (PLGA) nanoparticles carrying rapamycin, but not free rapamycin, are capable of inducing durable immunological tolerance to co-administered proteins that is characterized by the induction of tolerogenic dendritic cells, an increase in regulatory T cells, a reduction in B cell activation and germinal centre formation, and the inhibition of antigen-specific hypersensitivity reactions. Intravenous co-administration of tolerogenic nanoparticles with pegylated uricase inhibited the formation of ADAs in mice and non-human primates and normalized serum uric acid levels in uricase-deficient mice. Similarly, the subcutaneous co-administration of nanoparticles with adalimumab resulted in the durable inhibition of ADAs, leading to normalized pharmacokinetics of the anti-TNFα antibody and protection against arthritis in TNFα transgenic mice. Adjunct therapy with tolerogenic nanoparticles represents a novel and broadly applicable approach to prevent the formation of ADAs against biologic therapies.

Regulatory, biosafety and safety challenges for novel cells as substrates for human vaccines.

PubMed

Hess, Ralf D; Weber, Friedemann; Watson, Keith; Schmitt, Siegfried

2012-04-05

In the development of novel substrates used for production of human vaccines there has been significant progress made in recent years. Emerging and re-emerging infectious diseases like the recent porcine Influenza A virus (H1N1) pandemic necessitated the availability of unprecedented amounts of vaccines. In addition, the high demand for vaccines in the industrialised countries has also been paralleled by a steep increase in demand in developing countries. The manufacturing capability for viral vaccines produced in embryonated hen eggs and conventional/classical cell substrates, such as chicken embryo fibroblasts, has now reached its capacity limit. This constraint may be overcome by utilising other recognised cell substrates such as Madin Darby Canine Kidney (MDCK) (dog origin), Chinese Hamster Ovary (CHO) (hamster cells) or Vero cells (monkey origin) or as an alternative, introduce new cell substrates of human or avian origin. Using new cell substrates may prove to be a highly replication-proficient way of producing live viral vaccines such as Influenza A viruses. Despite some advantages, cell substrates may pose a small residual risk to humans since some of them are known to be tumourigenic in immunosuppressed animals. However, this residual risk should be considered acceptable by regulators. Safety testing requirements for cell substrates used in the manufacture of vaccines is mandated by published guidance from organisations such as World Health Organization (WHO), United States Food and Drug Administration (FDA), European Medicines Agency (EMA) and International Conferences on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human use (ICH) as well as requirements laid down in compendial monographs (Ph. Eur. and USP). This paper considers the guidance contained in these regulatory documents. In addition, the safety challenges and almost arbitrary risk-based classification of cell substrates used in the production of human vaccines together with compliance to GCCP (Good Cell Culture Practice) are discussed. Even though there has been tremendous progress in the last few years, reflected mainly by revisions and updates to regulatory guidance documents, there still is still no consensus between regulators nor significant harmonisation of the guidance documents or monographs. Copyright © 2012 Elsevier Ltd. All rights reserved.

Nonclinical Safety Assessment of Anti-Factor D: Key Strategies and Challenges for the Nonclinical Development of Intravitreal Biologics.

PubMed

Bantseev, Vladimir; Erickson, Rebecca; Leipold, Douglas; Amaya, Caroline; Miller, Paul E; Booler, Helen; Thackaberry, Evan A

The nonclinical toxicology program described here was designed to characterize the safety profile of anti-factor D (AFD; FCFD4514S, lampalizumab) to support intravitreal (ITV) administration in patients with geographic atrophy (GA). The toxicity of AFD was assessed in a single-dose and 6-month repeat-dose study in monkeys at doses up to 10 mg/eye. Toxicity was assessed by clinical ophthalmic examinations, intraocular pressure measurements, ocular photography, electroretinography, fluorescein angiography, optical coherence tomography, and anatomic pathology. Systemic exposure to AFD generally increased with the increase in dose level. The increases in mean maximal concentration and area under the curve values were roughly dose proportional. No accumulation of AFD was observed following 10 doses, and drug exposures were not affected by anti-drug antibodies. AFD was locally and systemically well tolerated in monkeys following ITV doses of up to 10 mg/eye. Ocular effects associated with AFD were limited to transient, reversible, dose-related, aqueous cell responses and injection-related, mild, vitreal cell responses. In the 6-month repeat-dose study, 2 monkeys had a nonspecific immune response to AFD that resulted in severe ocular inflammation, attributed to administration of a heterologous (humanized) protein. The comprehensive toxicology program in monkeys described here was designed to evaluate the safety profile of AFD and to support multiple ITV injections in the clinic. Administration of a heterologous (humanized) protein presents a challenge, and immunogenicity in nonclinical species is not predictive of immunogenicity in humans. Taken together, the results of the nonclinical program described here support the use of AFD in patients with GA.

CTLA4 aptamer delivers STAT3 siRNA to tumor-associated and malignant T cells

PubMed Central

Herrmann, Andreas; Priceman, Saul J.; Kujawski, Maciej; Xin, Hong; Cherryholmes, Gregory A.; Zhang, Wang; Zhang, Chunyan; Lahtz, Christoph; Kowolik, Claudia; Forman, Steve J.; Kortylewski, Marcin; Yu, Hua

2014-01-01

Intracellular therapeutic targets that define tumor immunosuppression in both tumor cells and T cells remain intractable. Here, we have shown that administration of a covalently linked siRNA to an aptamer (apt) that selectively binds cytotoxic T lymphocyte–associated antigen 4 (CTLA4apt) allows gene silencing in exhausted CD8+ T cells and Tregs in tumors as well as CTLA4-expressing malignant T cells. CTLA4 expression was upregulated in CD8+ T cells in the tumor milieu; therefore, CTLA4apt fused to a STAT3-targeting siRNA (CTLA4apt–STAT3 siRNA) resulted in internalization into tumor-associated CD8+ T cells and silencing of STAT3, which activated tumor antigen–specific T cells in murine models. Both local and systemic administration of CTLA4apt–STAT3 siRNA dramatically reduced tumor-associated Tregs. Furthermore, CTLA4apt–STAT3 siRNA potently inhibited tumor growth and metastasis in various mouse tumor models. Importantly, CTLA4 expression is observed in T cells of patients with blood malignancies, and CTLA4apt–STAT3 siRNA treatment of immunodeficient mice bearing human T cell lymphomas promoted tumor cell apoptosis and tumor growth inhibition. These data demonstrate that a CTLA4apt-based siRNA delivery strategy allows gene silencing in both tumor-associated T cells and tumor cells and inhibits tumor growth and metastasis. PMID:24892807

Recapitulation of spinal motor neuron-specific disease phenotypes in a human cell model of spinal muscular atrophy

PubMed Central

Wang, Zhi-Bo; Zhang, Xiaoqing; Li, Xue-Jun

2013-01-01

Establishing human cell models of spinal muscular atrophy (SMA) to mimic motor neuron-specific phenotypes holds the key to understanding the pathogenesis of this devastating disease. Here, we developed a closely representative cell model of SMA by knocking down the disease-determining gene, survival motor neuron (SMN), in human embryonic stem cells (hESCs). Our study with this cell model demonstrated that knocking down of SMN does not interfere with neural induction or the initial specification of spinal motor neurons. Notably, the axonal outgrowth of spinal motor neurons was significantly impaired and these disease-mimicking neurons subsequently degenerated. Furthermore, these disease phenotypes were caused by SMN-full length (SMN-FL) but not SMN-Δ7 (lacking exon 7) knockdown, and were specific to spinal motor neurons. Restoring the expression of SMN-FL completely ameliorated all of the disease phenotypes, including specific axonal defects and motor neuron loss. Finally, knockdown of SMN-FL led to excessive mitochondrial oxidative stress in human motor neuron progenitors. The involvement of oxidative stress in the degeneration of spinal motor neurons in the SMA cell model was further confirmed by the administration of N-acetylcysteine, a potent antioxidant, which prevented disease-related apoptosis and subsequent motor neuron death. Thus, we report here the successful establishment of an hESC-based SMA model, which exhibits disease gene isoform specificity, cell type specificity, and phenotype reversibility. Our model provides a unique paradigm for studying how motor neurons specifically degenerate and highlights the potential importance of antioxidants for the treatment of SMA. PMID:23208423

Characterization of human-induced pluripotent stem cell-derived cardiomyocytes: bioenergetics and utilization in safety screening.

PubMed

Rana, Payal; Anson, Blake; Engle, Sandra; Will, Yvonne

2012-11-01

Cardiotoxicity remains the number one reason for drug withdrawal from the market, and Food and Drug Administration issued black box warnings, thus demonstrating the need for more predictive preclinical safety screening, especially early in the drug discovery process when much chemical substrate is available. Whereas human-ether-a-go-go related gene screening has become routine to mitigate proarrhythmic risk, the development of in vitro assays predicting additional on- and off-target biochemical toxicities will benefit from cellular models exhibiting true cardiomyocyte characteristics such as native tissue-like mitochondrial activity. Human stem cell-derived tissue cells may provide such a model. This hypothesis was tested using a combination of flux analysis, gene and protein expression, and toxicity-profiling techniques to characterize mitochondrial function in induced pluripotent stem cell (iPSC) derived human cardiomyocytes in the presence of differing carbon sources over extended periods in cell culture. Functional analyses demonstrate that iPSC-derived cardiomyocytes are (1) capable of utilizing anaerobic or aerobic respiration depending upon the available carbon substrate and (2) bioenergetically closest to adult heart tissue cells when cultured in galactose or galactose supplemented with fatty acids. We utilized this model to test a variety of kinase inhibitors with known clinical cardiac liabilities for their potential toxicity toward these cells. We found that the kinase inhibitors showed a dose-dependent toxicity to iPSC cardiomyocytes grown in galactose and that oxygen consumption rates were significantly more affected than adenosine triphosphate production. Sorafenib was found to have the most effect, followed by sunitinib, dasatinib, imatinib, lapatinib, and nioltinib.

JAK inhibition alleviates the cellular senescence-associated secretory phenotype and frailty in old age

PubMed Central

Xu, Ming; Tchkonia, Tamara; Ding, Husheng; Ogrodnik, Mikolaj; Lubbers, Ellen R.; Pirtskhalava, Tamar; White, Thomas A.; Johnson, Kurt O.; Stout, Michael B.; Mezera, Vojtech; Giorgadze, Nino; Jensen, Michael D.; LeBrasseur, Nathan K.; Kirkland, James L.

2015-01-01

Chronic, low grade, sterile inflammation frequently accompanies aging and age-related diseases. Cellular senescence is associated with the production of proinflammatory chemokines, cytokines, and extracellular matrix (ECM) remodeling proteases, which comprise the senescence-associated secretory phenotype (SASP). We found a higher burden of senescent cells in adipose tissue with aging. Senescent human primary preadipocytes as well as human umbilical vein endothelial cells (HUVECs) developed a SASP that could be suppressed by targeting the JAK pathway using RNAi or JAK inhibitors. Conditioned medium (CM) from senescent human preadipocytes induced macrophage migration in vitro and inflammation in healthy adipose tissue and preadipocytes. When the senescent cells from which CM was derived had been treated with JAK inhibitors, the resulting CM was much less proinflammatory. The administration of JAK inhibitor to aged mice for 10 wk alleviated both adipose tissue and systemic inflammation and enhanced physical function. Our findings are consistent with a possible contribution of senescent cells and the SASP to age-related inflammation and frailty. We speculate that SASP inhibition by JAK inhibitors may contribute to alleviating frailty. Targeting the JAK pathway holds promise for treating age-related dysfunction. PMID:26578790

Dose- and Time-Dependent Response of Human Leukemia (HL-60) Cells to Arsenic Trioxide Treatment

PubMed Central

Yedjou, Clement G.; Moore, Pamela; Tchounwou, Paul B.

2006-01-01

The treatment of acute promyelocytic leukemia (APL) has been based on the administration of all-trans retinoic acid plus anthracycline chemotherapy, which is very effective as first line therapy; however 25 to 30% of patients will relapse with their disease becoming refractory to conventional therapy. Recently, studies have shown arsenic trioxide to be effective in the treatment of acute promyelocytic leukemia. In this study, we used the human leukemia (HL-60) cell line as a model to evaluate the cytoxicity of arsenic trioxide based on the MTT assay. Data obtained from this assay indicated that arsenic trioxide significantly reduced the viability of HL-60 cells, showing LD50 values of 14.26 ± 0.5μg/mL, 12.54 ± 0.3μg/mL, and 6.4 ± 0.6μg/mL upon 6, 12, and 24 hours of exposure, respectively; indicating a dose- and time-dependent response relationship. Findings from the present study indicate that arsenic trioxide is highly cytotoxic to human leukemia (HL-60) cells, supporting its use as an effective therapeutic agent in the management of acute promyelocytic leukemia. PMID:16823087

Improved efficacy of allergen-specific immunotherapy by JAK inhibition in a murine model of allergic asthma

PubMed Central

Alessandrini, Francesca; Fuchs, Helmut; Gailus-Durner, Valerie; Hrabě de Angelis, Martin; Russkamp, Dennis; Chaker, Adam; Ollert, Markus; Gutermuth, Jan; Schmidt-Weber, Carsten B.

2017-01-01

Background Allergen-specific immunotherapy (AIT) is the only curative treatment for type-1 allergies, but sometimes shows limited therapeutic response as well as local and systemic side effects. Limited control of local inflammation and patient symptoms hampers its widespread use in severe allergic asthma. Objective Our aim was to evaluate whether AIT is more effective in suppression of local inflammation if performed under the umbrella of short-term non-specific immunomodulation using a small molecule inhibitor of JAK pathways. Methods In C57BL/6J mice, a model of ovalbumin (OVA)-induced allergic airway inflammation and allergen-specific immunotherapy was combined with the administration of Tofacitinib (TOFA, a FDA-approved JAK inhibitor) from 48 hours prior to 48 hours after therapeutic OVA-injection. The effect of TOFA on human FOXP3+CD4+ T cells was studied in vitro. Results AIT combined with short-term TOFA administration was significantly more effective in suppressing total cell and eosinophil infiltration into the lung, local cytokine production including IL-1β and CXCL1 and showed a trend for the reduction of IL-4, IL-13, TNF-α and IL-6 compared to AIT alone. Furthermore, TOFA co-administration significantly reduced systemic IL-6, IL-1β and OVA-specific IgE levels and induced IgG1 to the same extent as AIT alone. Additionally, TOFA enhanced the induction of human FOXP3+CD4+ T cells. Conclusions This proof of concept study shows that JAK inhibition did not inhibit tolerance induction, but improved experimental AIT at the level of local inflammation. The improved control of local inflammation might extend the use of AIT in more severe conditions such as polyallergy, asthma and high-risk patients suffering from mastocytosis or anaphylaxis. PMID:28570653

Improved efficacy of allergen-specific immunotherapy by JAK inhibition in a murine model of allergic asthma.

PubMed

Aguilar-Pimentel, Antonio; Graessel, Anke; Alessandrini, Francesca; Fuchs, Helmut; Gailus-Durner, Valerie; Hrabě de Angelis, Martin; Russkamp, Dennis; Chaker, Adam; Ollert, Markus; Blank, Simon; Gutermuth, Jan; Schmidt-Weber, Carsten B

2017-01-01

Allergen-specific immunotherapy (AIT) is the only curative treatment for type-1 allergies, but sometimes shows limited therapeutic response as well as local and systemic side effects. Limited control of local inflammation and patient symptoms hampers its widespread use in severe allergic asthma. Our aim was to evaluate whether AIT is more effective in suppression of local inflammation if performed under the umbrella of short-term non-specific immunomodulation using a small molecule inhibitor of JAK pathways. In C57BL/6J mice, a model of ovalbumin (OVA)-induced allergic airway inflammation and allergen-specific immunotherapy was combined with the administration of Tofacitinib (TOFA, a FDA-approved JAK inhibitor) from 48 hours prior to 48 hours after therapeutic OVA-injection. The effect of TOFA on human FOXP3+CD4+ T cells was studied in vitro. AIT combined with short-term TOFA administration was significantly more effective in suppressing total cell and eosinophil infiltration into the lung, local cytokine production including IL-1β and CXCL1 and showed a trend for the reduction of IL-4, IL-13, TNF-α and IL-6 compared to AIT alone. Furthermore, TOFA co-administration significantly reduced systemic IL-6, IL-1β and OVA-specific IgE levels and induced IgG1 to the same extent as AIT alone. Additionally, TOFA enhanced the induction of human FOXP3+CD4+ T cells. This proof of concept study shows that JAK inhibition did not inhibit tolerance induction, but improved experimental AIT at the level of local inflammation. The improved control of local inflammation might extend the use of AIT in more severe conditions such as polyallergy, asthma and high-risk patients suffering from mastocytosis or anaphylaxis.

Selective anti-tumor activity of the novel fluoropyrimidine polymer F10 towards G48a orthotopic GBM tumors.

PubMed

Gmeiner, William H; Lema-Tome, Carla; Gibo, Denise; Jennings-Gee, Jamie; Milligan, Carol; Debinski, Waldemar

2014-02-01

F10 is a novel anti-tumor agent with minimal systemic toxicity in vivo and which displays strong cytotoxicity towards glioblastoma (GBM) cells in vitro. Here we investigate the cytotoxicity of F10 towards GBM cells and evaluate the anti-tumor activity of locally-administered F10 towards an orthotopic xenograft model of GBM. The effects of F10 on thymidylate synthase (TS) inhibition and Topoisomerase 1 (Top1) cleavage complex formation were evaluated using TS activity assays and in vivo complex of enzyme bioassays. Cytotoxicity of F10 towards normal brain was evaluated using cortices from embryonic (day 18) mice. F10 displays minimal penetrance of the blood-brain barrier and was delivered by intra-cerebral (i.c.) administration and prospective anti-tumor response towards luciferase-expressing G48a human GBM tumors in nude mice was evaluated using IVIS imaging. Histological examination of tumor and normal brain tissue was used to assess the selectivity of anti-tumor activity. F10 is cytotoxic towards G48a, SNB-19, and U-251 MG GBM cells through dual targeting of TS and Top1. F10 is not toxic to murine primary neuronal cultures. F10 is well-tolerated upon i.c. administration and induces significant regression of G48a tumors that is dose-dependent. Histological analysis from F10-treated mice revealed tumors were essentially completely eradicated in F10-treated mice while vehicle-treated mice displayed substantial infiltration into normal tissue. F10 displays strong efficacy for GBM treatment with minimal toxicity upon i.c. administration establishing F10 as a promising drug-candidate for treating GBM in human patients.

Reduction of human anti-tetanus toxoid antibody in hu-PBL-SCID mice by immunodominant peptides of tetanus toxoid

PubMed Central

Jackson, D J; Elson, C J; Kumpel, B M

2004-01-01

Immunotherapy of murine autoimmune and allergic diseases by administration of peptides corresponding to the dominant T cell epitope is a reality. However, problems remain in applying this therapy to reduce antibody responses in humans. To overcome these difficulties, a preclinical system was developed to test the effect of immunodominant peptides from a common antigen, tetanus toxoid (TT), on the long-term human anti-TT response. Individuals whose T cells proliferated against dominant TT peptides were identified. Peripheral blood leucocytes (PBL) from these donors were injected intraperitoneally (i.p.) into mice with severe combined immunodeficiency (SCID) that had been depleted of murine natural killer (NK) cells (hu-PBL-SCID mice). Peptides or PBS were injected i.p. before a further injection of PBL and immunization with TT. The concentration of human IgG and anti-TT in murine plasma was followed for 10 weeks. The total IgG was similar in both groups. By contrast, there was a statistically significant reduction in IgG anti-TT from eight weeks onwards. It is considered that the hu-PBL-SCID model system may provide a means by which the efficacy of peptide immunotherapy for reduction of pathological antibodies in humans can be examined. PMID:15270840

Pluripotent stem cells in translation: a Food and Drug Administration-National Institutes of Health collaboration.

PubMed

Kleitman, Naomi; Rao, Mahendra S; Owens, David F

2013-07-01

Recently, the U.S. Food and Drug Administration (FDA), the U.S. National Institutes of Health, and the stem cell research community have collaborated on a series of workshops that address moving pluripotent stem cell therapies into the clinic. The first two workshops in the series focused on preclinical science, and a third, future workshop will focus on clinical trials. This summary addresses major points from both of the recent preclinically focused meetings. When entering into a therapeutics developmental program based on pluripotent cells, investigators must make decisions at the very early stages that will have major ramifications during later phases of development. Presentations and discussions from both invited participants and FDA staff described the need to characterize and document the quality, variability, and suitability of the cells and commercial reagents used at every translational stage. This requires consideration of future regulatory requirements, ranging from donor eligibility of the original source material to the late-stage manufacturing protocols. Federal, industrial, and academic participants agreed that planning backward is the best way to anticipate what evidence will be needed to justify human testing of novel therapeutics and to eliminate wasted efforts.

Pluripotent Stem Cells in Translation: A Food and Drug Administration-National Institutes of Health Collaboration

PubMed Central

Kleitman, Naomi; Owens, David F.

2013-01-01

Recently, the U.S. Food and Drug Administration (FDA), the U.S. National Institutes of Health, and the stem cell research community have collaborated on a series of workshops that address moving pluripotent stem cell therapies into the clinic. The first two workshops in the series focused on preclinical science, and a third, future workshop will focus on clinical trials. This summary addresses major points from both of the recent preclinically focused meetings. When entering into a therapeutics developmental program based on pluripotent cells, investigators must make decisions at the very early stages that will have major ramifications during later phases of development. Presentations and discussions from both invited participants and FDA staff described the need to characterize and document the quality, variability, and suitability of the cells and commercial reagents used at every translational stage. This requires consideration of future regulatory requirements, ranging from donor eligibility of the original source material to the late-stage manufacturing protocols. Federal, industrial, and academic participants agreed that planning backward is the best way to anticipate what evidence will be needed to justify human testing of novel therapeutics and to eliminate wasted efforts. PMID:23757505

Structurally well-defined macrophage activating factor derived from vitamin D3-binding protein has a potent adjuvant activity for immunization.

PubMed

Yamamoto, N; Naraparaju, V R

1998-06-01

Freund's adjuvant produced severe inflammation that augments development of antibodies. Thus, mixed administration of antigens with adjuvant was not required as long as inflammation was induced in the hosts. Since macrophage activation for phagocytosis and antigen processing is the first step of antibody development, inflammation-primed macrophage activation plays a major role in immune development. Therefore, macrophage activating factor should act as an adjuvant for immunization. The inflammation-primed macrophage activation process is the major macrophage activating cascade that requires participation of serum vitamin D3-binding protein (DBP; human DBP is known as Gc protein) and glycosidases of B and T lymphocytes. Stepwise incubation of Gc protein with immobilized beta-galactosidase and sialidase efficiently generated the most potent macrophage activating factor (designated GcMAF) we have ever encountered. Administration of GcMAF (20 or 100 pg/mouse) resulted in stimulation of the progenitor cells for extensive mitogenesis and activation of macrophages. Administration of GcMAF (100 pg/mouse) along with immunization of mice with sheep red blood cells (SRBC) produced a large number of anti-SRBC antibody secreting splenic cells in 2-4 days. Thus, GcMAF has a potent adjuvant activity for immunization. Although malignant tumours are poorly immunogenic, 4 days after GcMAF-primed immunization of mice with heat-killed Ehrlich ascites tumour cells, the ascites tumour was no longer transplantable in these mice.

Fluorescence contrast-enhanced proliferative lesion imaging by enema administration of indocyanine green in a rat model of colon carcinogenesis

PubMed Central

Onda, Nobuhiko; Mizutani-Morita, Reiko; Yamashita, Susumu; Nagahara, Rei; Matsumoto, Shinya; Yoshida, Toshinori; Shibutani, Makoto

2017-01-01

The fluorescent contrast agent indocyanine green (ICG) is approved by the Food and Drug Administration for clinical applications. We previously reported that cultured human colon tumor cells preferentially take up ICG by endocytic activity in association with disruption of their tight junctions. The present study explored ICG availability in fluorescence imaging of the colon to identify proliferative lesions during colonoscopy. The cellular uptake of ICG in cultured rat colon tumor cells was examined using live-cell imaging. Colon lesions in rats administered an ICG-containing enema were further assessed in rats with azoxymethane-induced colon carcinogenesis, using in vivo endoscopy, ex vivo microscopy, and immunofluorescence microscopy. The uptake of ICG by the cultured cells was temperature-dependent. The intracellular retention of the dye in the membrane trafficking system suggested endocytosis as the uptake mechanism. ICG administered via enema accumulated in colon proliferative lesions ranging from tiny aberrant crypt foci to adenomas and localized in proliferating cells. Fluorescence endoscopy detected these ICG-positive colonic proliferative lesions in vivo. The immunoreactivity of the tight-junction molecule occludin was altered in the proliferative lesions, suggesting the disruption of the integrity of tight junctions. These results suggest that fluorescence contrast-enhanced imaging following the administration of an ICG-containing enema can enhance the detection of mucosal proliferative lesions of the colon during colonoscopy. The tissue preference of ICG in the rat model evaluated in this study can be attributed to the disruption of tight junctions, which in turn promotes endocytosis by proliferative cells and the cellular uptake of ICG. PMID:29163827

New evidence for the therapeutic potential of curcumin to treat nonalcoholic fatty liver disease in humans.

PubMed

Inzaugarat, María Eugenia; De Matteo, Elena; Baz, Placida; Lucero, Diego; García, Cecilia Claudia; Gonzalez Ballerga, Esteban; Daruich, Jorge; Sorda, Juan Antonio; Wald, Miriam Ruth; Cherñavsky, Alejandra Claudia

2017-01-01

The immune system acts on different metabolic tissues that are implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Leptin and linoleic acid have the ability to potentially affect immune cells, whereas curcumin is a known natural polyphenol with antioxidant and anti-inflammatory properties. This study was designed to evaluate the pro-inflammatory and pro-oxidant effects of leptin and linoleic acid on immune cells from patients with NAFLD and to corroborate the modulatory effects of curcumin and its preventive properties against the progression of NAFLD using a high-fat diet (HFD)-induced NAFLD/nonalcoholic steatohepatitis mouse model. The ex vivo experiments showed that linoleic acid increased the production of reactive oxygen species in monocytes and liver macrophages, whereas leptin enhanced tumor necrosis factor-α (TNF-α) production in monocytes and interferon-γ production in circulating CD4+ cells. Conversely, oral administration of curcumin prevented HFD-induced liver injury, metabolic alterations, intrahepatic CD4+ cell accumulation and the linoleic acid- and leptin- induced pro-inflammatory and pro-oxidant effects on mouse liver macrophages. Our findings provide new evidence for the therapeutic potential of curcumin to treat human NAFLD. However, the development of a preventive treatment targeting human circulating monocytes and liver macrophages as well as peripheral and hepatic CD4+ cells requires additional research.

Edaravone Protects against Methylglyoxal-Induced Barrier Damage in Human Brain Endothelial Cells

PubMed Central

Tóth, Andrea E.; Walter, Fruzsina R.; Bocsik, Alexandra; Sántha, Petra; Veszelka, Szilvia; Nagy, Lajos; Puskás, László G.; Couraud, Pierre-Olivier; Takata, Fuyuko; Dohgu, Shinya; Kataoka, Yasufumi; Deli, Mária A.

2014-01-01

Background Elevated level of reactive carbonyl species, such as methylglyoxal, triggers carbonyl stress and activates a series of inflammatory responses leading to accelerated vascular damage. Edaravone is the active substance of a Japanese medicine, which aids neurological recovery following acute brain ischemia and subsequent cerebral infarction. Our aim was to test whether edaravone can exert a protective effect on the barrier properties of human brain endothelial cells (hCMEC/D3 cell line) treated with methylglyoxal. Methodology Cell viability was monitored in real-time by impedance-based cell electronic sensing. The barrier function of the monolayer was characterized by measurement of resistance and flux of permeability markers, and visualized by immunohistochemistry for claudin-5 and β-catenin. Cell morphology was also examined by holographic phase imaging. Principal Findings Methylglyoxal exerted a time- and dose-dependent toxicity on cultured human brain endothelial cells: a concentration of 600 µM resulted in about 50% toxicity, significantly reduced the integrity and increased the permeability of the barrier. The cell morphology also changed dramatically: the area of cells decreased, their optical height significantly increased. Edaravone (3 mM) provided a complete protection against the toxic effect of methylglyoxal. Co-administration of edaravone restored cell viability, barrier integrity and functions of brain endothelial cells. Similar protection was obtained by the well-known antiglycating molecule, aminoguanidine, our reference compound. Conclusion These results indicate for the first time that edaravone is protective in carbonyl stress induced barrier damage. Our data may contribute to the development of compounds to treat brain endothelial dysfunction in carbonyl stress related diseases. PMID:25033388

Silymarin inhibits melanoma cell growth both in vitro and in vivo by targeting cell cycle regulators, angiogenic biomarkers and induction of apoptosis.

PubMed

Vaid, Mudit; Singh, Tripti; Prasad, Ram; Katiyar, Santosh K

2015-11-01

Cutaneous malignant melanoma is the leading cause of death from skin diseases and is often associated with activating mutations of the proto-oncogene BRAF. To develop more effective strategies for the prevention or treatment of melanoma, we have examined the inhibitory effects of silymarin, a flavanoid from Silybum marianum, on melanoma cells. Using A375 (BRAF-mutated) and Hs294t (non BRAF-mutated but highly metastatic) human melanoma cell lines, we found that in vitro treatment with silymarin resulted in a dose-dependent: (i) reduction in cell viability; (ii) enhancement of either Go/G1 (A375) or G2-M (Hs294t) phase cell cycle arrest with corresponding alterations in cyclins and cyclin-dependent kinases; and (iii) induction of apoptosis. The silymarin-induced apoptosis of human melanoma cells was associated with a reduction in the levels of anti-apoptotic proteins (Bcl-2 and Bcl-xl), an increase in the levels of pro-apoptotic protein (Bax), and activation of caspases. Further, oral administration of silymarin (500 mg/kg body weight/2× a week) significantly inhibited (60%, P < 0.01) the growth of BRAF-mutated A375 melanoma tumor xenografts, and this was associated with: (i) inhibition of cell proliferation; (ii) induction of apoptosis of tumor cells; (iii) alterations in cell cycle regulatory proteins; and (iv) reduced expression of tumor angiogenic biomarkers in tumor xenograft tissues. These results indicate that silymarin may have a chemotherapeutic effect on human melanoma cell growth and warrant its further evaluation. © 2014 Wiley Periodicals, Inc.

Versatile graphene biosensors for enhancing human cell therapy.

PubMed

Vlăsceanu, George M; Amărandi, Roxana-Maria; Ioniță, Mariana; Tite, Teddy; Iovu, Horia; Pilan, Luisa; Burns, Jorge S

2018-05-01

Technological advances in engineering and cell biology stimulate novel approaches for medical treatment, in particular cell-based therapy. The first cell-based gene therapy against cancer was recently approved by the US Food and Drug Administration. Progress in cancer diagnosis includes a blood test detecting five cancer types. Numerous stem cell phase I/II clinical trials showing safety and efficacy will soon pursue qualifying criteria for advanced therapy medicinal products (ATMP), aspiring to join the first stem-cell therapy approved by the European Medicines Agency. Cell based therapy requires extensive preclinical characterisation of biomarkers indicating mechanisms of action crucial to the desired therapeutic effect. Quantitative analyses monitoring critical functions for the manufacture of optimal cell and tissue-based clinical products include successful potency assays for implementation. The challenge to achieve high quality measurement is increasingly met by progress in biosensor design. We adopt a cell therapy perspective to highlight recent examples of graphene-enhanced biointerfaces for measurement of biomarkers relevant to cancer treatment, diagnosis and tissue regeneration. Graphene based biosensor design problems can thwart their use for health care transformative point of care testing and real-time applications. We discuss concerns to be addressed and emerging solutions for establishing clinical grade biosensors to accelerate human cell therapy. Copyright © 2018 Elsevier B.V. All rights reserved.

Formononetin suppresses the proliferation of human non-small cell lung cancer through induction of cell cycle arrest and apoptosis.

PubMed

Yang, Yi; Zhao, Yi; Ai, Xinghao; Cheng, Baijun; Lu, Shun

2014-01-01

Formononetin is a novel herbal isoflavonoid isolated from Astragalus membranaceus and possesses antitumorigenic properties. In the present study, we investigated the anti-proliferative effects of formononetin on human non-small cell lung cancer (NSCLC), and further elucidated the molecular mechanism underlying the anti-tumor property. MTT assay showed that formononetin treatment significantly inhibited the proliferation of two NSCLC cell lines including A549 and NCI-H23 in a time- and dose-dependent manner. Flow cytometric analysis demonstrated that formononetin induced G1-phase cell cycle arrest and promoted cell apoptosis in NSCLC cells. On the molecular level, we observed that exposure to formononetin altered the expression levels of cell cycle arrest-associated proteins p21, cyclin A and cyclin D1. Meanwhile, the apoptosis-related proteins cleaved caspase-3, bax and bcl-2 were also changed following treatment with formononetin. In addition, the expression level of p53 was dose-dependently upregulated after administration with formononetin. We also found that formononetin treatment increased the phosphorylation of p53 at Ser15 and Ser20 and enhances its transcriptional activity in a dose-dependent manner. Collectively, these results demonstrated that formononetin might be a potential chemopreventive drug for lung cancer therapy through induction of cell cycle arrest and apoptosis in NSCLC cells.

A novel matrine derivate inhibits differentiated human hepatoma cells and hepatic cancer stem-like cells by suppressing PI3K/AKT signaling pathways

PubMed Central

Liu, Ying; Qi, Yang; Bai, Zhi-hui; Ni, Chen-xu; Ren, Qi-hui; Xu, Wei-heng; Xu, Jing; Hu, Hong-gang; Qiu, Lei; Li, Jian-zhong; He, Zhi-gao; Zhang, Jun-ping

2017-01-01

Matrine is an alkaloid extracted from a Chinese herb Sophora flavescens Ait, which has shown chemopreventive potential against various cancers. In this study, we evaluated the anticancer efficacy of a novel derivative of matrine, (6aS, 10S, 11aR, 11bR, 11cS)-10- methylamino-dodecahydro- 3a,7a-diazabenzo (de) (MASM), against human hepatocellular carcinoma (HCC) cells and their corresponding sphere cells in vitro and in vivo. Human HCC cell lines (Hep3B and Huh7) were treated with MASM. Cell proliferation was assessed using CCK8 and colony assays; cell apoptosis and cell cycle distributions were examined with flow cytometry. The expression of cell markers and signaling molecules was detected using Western blot and qRT-PCR analyses. A sphere culture technique was used to enrich cancer stem cells (CSC) in Hep3B and Huh7 cells. The in vivo antitumor efficacy of MASM was evaluated in Huh7 cell xenograft model in BALB/c nude mice, which were administered MASM (10 mg·kg−1·d−1, ig) for 3 weeks. After the treatment was completed, tumor were excised and weighed. A portion of tumor tissue was enzymatically dissociated to obtain a single cell suspension for the spheroid formation assays. MASM (2, 10, 20 μmol/L) dose-dependently inhibited the proliferation of HCC cells, and induced apoptosis, which correlated with a reduction in Bcl-2 expression and an increase in PARP cleavage. MASM also induced cell cycle arrest in G0/G1 phase, which was accompanied by increased p27 and decreased Cyclin D1 expression. Interestingly, MASM (2, 10, and 20 μmol/L) drastically reduced the EpCAM+/CD133+ cell numbers, suppressed the sphere formation, inhibited the expression of stem cell marker genes and promoted the expression of mature hepatocyte markers in the Hep3B and Huh7 spheroids. Additionally, MASM dose-dependently suppressed the PI3K/AKT/mTOR and AKT/GSK3β/β-catenin signaling pathways in Hep3B and Huh7 cells. In Huh7 xenograft bearing nude mice, MASM administration significantly inhibited Huh7 xenograft tumor growth and markedly reduced the number of surviving cancer stem-like cells in the tumors. MASM administration also reduced the expression of stem cell markers while increasing the expression of mature hepatocyte markers in the tumor tissues. The novel derivative of matrine, MASM, markedly suppresses HCC tumor growth through multiple mechanisms, and it may be a promising candidate drug for the treatment of hepatocellular carcinoma. PMID:27773936

CD44v6 expression in human skin keratinocytes as a possible mechanism for carcinogenesis associated with chronic arsenic exposure.

PubMed

Huang, S; Guo, S; Guo, F; Yang, Q; Xiao, X; Murata, M; Ohnishi, S; Kawanishi, S; Ma, N

2013-01-14

Inorganic arsenic is a well-known human skin carcinogen. Chronic arsenic exposure results in various types of human skin lesions, including squamous cell carcinoma (SCC). To investigate whether mutant stem cells participate in arsenic-associated carcinogenesis, we repeatedly exposed the HaCaT cells line to an environmentally relevant level of arsenic (0.05 ppm) in vitro for 18 weeks. Following sodium arsenic arsenite administration, cell cycle, colony-forming efficiency (CFE), cell tumorigenicity, and expression of CD44v6, NF-κB and p53, were analyzed at different time points (0, 5, 10, 15, 20, 25 and 30 passages). We found that a chronic exposure of HaCaT cells to a low level of arsenic induced a cancer stem- like phenotype. Furthermore, arsenic-treated HaCaT cells also became tumorigenic in nude mice, their growth cycle was predominantly in G2/M and S phases. Relative to nontreated cells, they exhibited a higher growth rate and a significant increase in CFE. Western blot analysis found that arsenic was capable of increasing cell proliferation and sprouting of cancer stem-like phenotype. Additionally, immunohistochemical analysis demonstrated that CD44v6 expression was up-regulated in HaCaT cells exposed to a low level of arsenic during early stages of induction. The expression of CD44v6 in arsenic-treated cells was positively correlated with their cloning efficiency in soft agar (r=0.949, P=0.01). Likewise, the expressions of activating transcription factor NF-κB and p53 genes in the arsenic-treated HaCaT cells were significantly higher than that in non-treated cells. Higher expressions of CD44v6, NF-κB and p53 were also observed in tumor tissues isolated from Balb/c nude mice. The present results suggest that CD44v6 may be a biomarker of arsenic-induced neoplastic transformation in human skin cells, and that arsenic promotes malignant transformation in human skin lesions through a NF-κB signaling pathway-stimulated expression of CD44v6.

Tumor-associated down-regulation of 15-lipoxygenase-1 is reversed by celecoxib in colorectal cancer.

PubMed

Heslin, Martin J; Hawkins, Ashley; Boedefeld, William; Arnoletti, J Pablo; Frolov, Andrey; Soong, Richie; Urist, Marshall M; Bland, Kirby I

2005-06-01

To evaluate the role of celecoxib on 15-lipoxygenase-1 (15-LOX-1) expression, protein levels, and rates of apoptosis in colorectal cancer cell lines. Also, to evaluate the expression of 15-LOX-1 in human normal mucosa, adenoma, and carcinoma with correlation to overall survival. The function of 15-LOX-1 is to maintain normal rates of apoptosis (programmed cell death). Decreased apoptosis is one mechanism of cancer growth and dissemination. It is our hypothesis that expression of 15-LOX-1 is reduced in human colorectal cancer (CRC) and the administration of celecoxib can reverse this process and induce apoptosis. Effect of celecoxib in cell culture: The effect of 40 micromol/L celecoxib was compared with untreated controls in tissue culture utilizing HT-29 and DLD-1 CRC cell lines. Expression of 15-LOX-1 protein was measured by immunoblot. Induction of apoptosis was evaluated by annexin V staining. All data are presented as mean +/- SEM, with significance defined as P < 0.05. 15-LOX-1 in human CRC: From February 1998 to January 2002, 126 patients underwent surgical resection of either colorectal adenomas (n = 24) or carcinomas (n = 102), or both (n = 25). Tissue was macrodissected, snap frozen, and stored at -80 degrees C. After tissue processing, RNA was extracted and gene expression of 15-LOX-1 was quantified utilizing ABI prism real-time quantitative RT-PCR. Significance evaluated by the Wilcoxon signed rank test. Effect of celecoxib in cell culture: After 72 hours of treatment with celecoxib, immunoblot demonstrated a 1.5- to 2-fold increase in 15-LOX-1 protein expression in HT-29 and DLD-1 cells, respectively. Celecoxib produced greater than a 2-fold increase in the rate of apoptosis compared with control cells in both cell lines (P < 0.05). 15-LOX-1 in human CRC: The mean age of the patients was 62 +/- 1 years; 78% were white and 48% were female. The mean size of the polyps and cancers were 3.0 +/- 0.4 and 5.0 +/- 0.1 cm, respectively. Expression of 15-LOX-1 relative to S9 was 30 in normal mucosa and significantly down-regulated to 11 in adenomas and 16 in carcinomas (P < 0.05). 15-LOX-1 gene expression is significantly reduced in both human colorectal adenomas and carcinomas and associated with decreased survival. Administration of celecoxib restores 15-LOX-1 protein expression and induces apoptosis. Down-regulation of 15-LOX-1 is an early event in the adenoma to carcinoma sequence, and reversal with celecoxib may represent one mechanism for chemoprevention of polyps or treatment of carcinomas.

Hyaluronic acid increases tendon derived cell viability and proliferation in vitro: comparative study of two different hyaluronic acid preparations by molecular weight.

PubMed

Gallorini, Marialucia; Berardi, Anna C; Berardocco, Martina; Gissi, Clarissa; Maffulli, Nicola; Cataldi, Amelia; Oliva, Francesco

2017-01-01

Hyaluronic Acid (HA) has been already approved by Food and Drug Administration (FDA) for osteoarthritis (OA), while its use in the treatment of tendinopathy is still debated. The aim of this study was to evaluate the effects of two different HA on human rotator cuff tendon derived cells in terms of cell viability, proliferation and apoptosis. An in vitro model was developed on human tendon derived cells from rotator cuff tears to study the effects of two different HA preparations: Sinovial HL® (High-Low molecular weight) (MW: 80-100 kDa) and KDa Sinovial Forte SF (MW: 800-1200), at various concentrations. Tendon derived cells morphology was evaluated after 0, 7 and 14 d of culture. Viability and proliferation were analyzed after 0, 24, and 48 h of culture and apoptosis occurrence was assessed after 24 h of culture. All the HAPs tested here increased viability and proliferation, in a dose-dependent manner and they reduced apoptosis at early stages (24 h) compared to control cells (without HAPs). HAPs enhanced viability and proliferation and counteracted apoptosis in tendon derived cells.

Human Albumin Prevents 6-Hydroxydopamine-Induced Loss of Tyrosine Hydroxylase in In Vitro and In Vivo

PubMed Central

Zhang, Li-Juan; Xue, Yue-Qiang; Yang, Chun; Yang, Wei-Hua; Chen, Long; Zhang, Qian-Jin; Qu, Ting-Yu; Huang, Shile; Zhao, Li-Ru; Wang, Xiao-Min; Duan, Wei-Ming

2012-01-01

Human albumin has recently been demonstrated to protect brain neurons from injury in rat ischemic brain. However, there is no information available about whether human albumin can prevent loss of tyrosine hydroxylase (TH) expression of dopaminergic (DA) neurons induced by 6-hydroxydopamine (6-OHDA) toxicity that is most commonly used to create a rat model of Parkinson's disease (PD). In the present study, two microliters of 1.25% human albumin were stereotaxically injected into the right striatum of rats one day before or 7 days after the 6-OHDA lesion in the same side. D-Amphetamine-induced rotational asymmetry was measured 7 days, 3 and 10 weeks after 6-OHDA lesion. We observed that intrastriatal administration of human albumin significantly reduced the degree of rotational asymmetry. The number of TH-immunoreactive neurons present in the substantia nigra was greater in 6-OHDA lesioned rats following human albumin-treatment than non-human albumin treatment. TH-immunoreactivity in the 6-OHDA-lesioned striatum was also significantly increased in the human albumin-treated rats. To examine the mechanisms underlying the effects of human albumin, we challenged PC12 cells with 6-OHDA as an in vitro model of PD. Incubation with human albumin prevented 6-OHDA-induced reduction of cell viability in PC12 cell cultures, as measured by MTT assay. Furthermore, human albumin reduced 6-OHDA-induced formation of reactive oxygen species (ROS) and apoptosis in cultured PC12 cells, as assessed by flow cytometry. Western blot analysis showed that human albumin inhibited 6-OHDA-induced activation of JNK, c-Jun, ERK, and p38 mitogen-activated protein kinases (MAPK) signaling in PC12 cultures challenged with 6-OHDA. Human albumin may protect against 6-OHDA toxicity by influencing MAPK pathway followed by anti-ROS formation and anti-apoptosis. PMID:22815976

Convection enhanced delivery of panobinostat (LBH589)-loaded pluronic nano-micelles prolongs survival in the F98 rat glioma model

PubMed Central

Singleton, WG; Collins, AM; Bienemann, AS; Killick-Cole, CL; Haynes, HR; Asby, DJ; Butts, CP; Wyatt, MJ; Barua, NU; Gill, SS

2017-01-01

Background The pan-histone deacetylase inhibitor panobinostat is a potential therapy for malignant glioma, but it is water insoluble and does not cross the blood–brain barrier when administered systemically. In this article, we describe the in vitro and in vivo efficacy of a novel water-soluble nano-micellar formulation of panobinostat designed for administration by convection enhanced delivery (CED). Materials and methods The in vitro efficacy of panobinostat-loaded nano-micelles against rat F98, human U87-MG and M059K glioma cells and against patient-derived glioma stem cells was measured using a cell viability assay. Nano-micelle distribution in rat brain was analyzed following acute CED using rhodamine-labeled nano-micelles, and toxicity was assayed using immunofluorescent microscopy and synaptophysin enzyme-linked immunosorbent assay. We compared the survival of the bioluminescent syngenic F98/Fischer344 rat glioblastoma model treated by acute CED of panobinostat-loaded nano-micelles with that of untreated and vehicle-only-treated controls. Results Nano-micellar panobinostat is cytotoxic to rat and human glioma cells in vitro in a dose-dependent manner following short-time exposure to drug. Fluorescent rhodamine-labelled nano-micelles distribute with a volume of infusion/volume of distribution (Vi/Vd) ratio of four and five respectively after administration by CED. Administration was not associated with any toxicity when compared to controls. CED of panobinostat-loaded nano-micelles was associated with significantly improved survival when compared to controls (n=8 per group; log-rank test, P<0.001). One hundred percent of treated animals survived the 60-day experimental period and had tumour response on post-mortem histological examination. Conclusion CED of nano-micellar panobinostat represents a potential novel therapeutic option for malignant glioma and warrants translation into the clinic. PMID:28260886

Carbon nanotubes toxicology and effects on metabolism and immunological modification in vitro and in vivo

NASA Astrophysics Data System (ADS)

Chiaretti, M.; Mazzanti, G.; Bosco, S.; Bellucci, S.; Cucina, A.; LeFoche, F.; Carru, G. A.; Mastrangelo, S.; Di Sotto, A.; Masciangelo, R.; Chiaretti, A. M.; Balasubramanian, C.; DeBellis, G.; Micciulla, F.; Porta, N.; Deriu, G.; Tiberia, A.

2008-11-01

The aim of this research is focused on the biological effects of multi wall carbon nanotubes (MWCNTs) on three different human cell types, laboratory animals in vivo, and immunological effects. Large numbers of researchers are directly involved in the handling of nanostructured materials such as MWCNTs and nanoparticles. It is important to assess the potential health risks related to their daily exposure to carbon nanotubes. The administration of sterilized nanosamples has been performed on laboratory animals, in both acute and chronic administration, and the pathological effects on the parenchymal tissues have been investigated. We studied the serum immunological modifications after intraperitoneal administration of the MWCNTs. We did not observe any antigenic reaction; the screening of ANA, anti-ENA, anti-cardiolipin, C-ANCA and P-ANCA was negative. No quantitative modification of immunoglobulins was observed, hence no modification of humoral immunity was documented. We also studied the effects of MWCNTs on the proliferation of three different cell types. MCF-7 showed a significant inhibition of proliferation for all conditions studied, whereas hSMCs demonstrated a reduction of cell growth only for the highest MWCNTs concentrations after 72 h. Also, no growth modification was observed in the Caco-2 cell line. We observed that a low quantity of MWCNTs does not provoke any inflammatory reaction. However, for future medical applications, it is important to realize prosthesis based on MWCNTs, through studying the corresponding implantation effects. Moreover, it has to be emphasized that this investigation does not address, at the moment, the carcinogenicity of MWCNTs, which requires a detailed follow-up investigation on the specific topic. In view of the subsequent and more extensive use of MWCNTs, especially in applications where carbon nanotubes are injected into the human body for drug delivery, as a contrast agent carrying entities for MRI, or as the basic material of a new prosthesis generation, more extended tests and experiments are necessary.

Synthesis and plasma pharmacokinetics in CD-1 mice of a 18β-glycyrrhetinic acid derivative displaying anti-cancer activity.

PubMed

Lallemand, Benjamin; Ouedraogo, Moustapha; Wauthoz, Nathalie; Lamkami, Touria; Mathieu, Veronique; Jabin, Ivan; Amighi, Karim; Kiss, Robert; Dubois, Jacques; Goole, Jonathan

2013-03-01

The plasma pharmacokinetic profile in CD-1 mice of a novel 18β-glycyrrhetinic acid (GA) derivative, which displays in vitro anti-cancer activity, was assessed. This study involved an original one-step synthesis of N-(2-{3-[3,5-bis(trifluoromethyl)phenyl]ureido}ethyl)-glycyrrhetinamide, (2) a compound that displays marked anti-proteasome and anti-kinase activity. The bioselectivity profile of 2 on human normal NHDF fibroblasts vs human U373 glioblastoma cells was assessed. Maximal tolerated dose (MTD) profiling of 2 was carried out in CD1 mice, and its serum pharmacokinetics were profiled using an acute intravenous administration of 40 mg/kg body weight. Compound 2 displayed IC(50) in vitro growth inhibitory concentrations of 29 and 8 μm on NHDF fibroblasts and U373 glioblastoma cells, respectively, thus a bioselectivity index of ∼4. The intravenous pharmacokinetic parameters revealed that 2 was rapidly distributed (t(1/2dist) of ∼3 min) but slowly eliminated (t(1/2elim)  = ∼77 min). This study describes an original and reliable nanoemulsion of a GA derivative with both anti-proteasome and anti-kinase properties and that should be further tested in vivo using various human xenograft or murine syngeneic tumour models with both single and chronic intravenous administration. © 2012 The Authors. JPP © 2012. Royal Pharmaceutical Society.

α-Mangostin: a dietary antioxidant derived from the pericarp of Garcinia mangostana L. inhibits pancreatic tumor growth in xenograft mouse model.

PubMed

Hafeez, Bilal Bin; Mustafa, Ala; Fischer, Joseph W; Singh, Ashok; Zhong, Weixiong; Shekhani, Mohammed Ozair; Meske, Louise; Havighurst, Thomas; Kim, KyungMann; Verma, Ajit Kumar

2014-08-10

Pancreatic cancer (PC) is the most aggressive malignant disease, ranking as the fourth most leading cause of cancer-related death among men and women in the United States. In this study, we provide evidence of chemotherapeutic effects of α-mangostin, a dietary antioxidant isolated from the pericarp of Garcinia mangostana L. against human PC. The chemotherapeutic effect of α-mangostin was determined using four human PC cells (PL-45, PANC1, BxPC3, and ASPC1). α-Mangostin resulted in a significant inhibition of PC cells viability without having any effects on normal human pancreatic duct epithelial cells. α-Mangostin showed a dose-dependent increase of apoptosis in PC cells. Also, α-mangostin inhibited the expression levels of pNF-κB/p65Ser552, pStat3Ser727, and pStat3Tyr705. α-Mangostin inhibited DNA binding activity of nuclear factor kappa B (NF-κB) and signal transducer and activator 3 (Stat3). α-Mangostin inhibited the expression levels of matrix metallopeptidase 9 (MMP9), cyclin D1, and gp130; however, increased expression of tissue inhibitor of metalloproteinase 1 (TIMP1) was observed in PC cells. In addition, i.p. administration of α-mangostin (6 mg/kg body weight, 5 days a week) resulted in a significant inhibition of both primary (PL-45) and secondary (ASPC1) human PC cell-derived orthotopic and ectopic xenograft tumors in athymic nude mice. No sign of toxicity was observed in any of the mice administered with α-mangostin. α-Mangostin treatment inhibited the biomarkers of cell proliferation (Ki-67 and proliferating cell nuclear antigen [PCNA]) in the xenograft tumor tissues. We present, for the first time, that dietary antioxidant α-mangostin inhibits the growth of PC cells in vitro and in vivo. These results suggest the potential therapeutic efficacy of α-mangostin against human PC.

Enhanced anticancer effects of a mixture of low-dose mushrooms and Panax ginseng root extracts in human colorectal cancer cells.

PubMed

Lee, Mi So; Kim, Mi-Sook; Yoo, Jae Kuk; Lee, Ji Young; Ju, Jae Eun; Jeong, Youn Kyoung

2017-09-01

Worldwide, colorectal cancer is the third most common cancer in men and the second most common in women. As conventional colorectal cancer therapies result in various side effects, there is a need for adjuvant therapy that can enhance the conventional therapies without complications. In this study, we investigated the anticancer effects of combined mixture of the several medicinal mushrooms and Panax ginseng root extracts (also called Amex7) as an adjuvant compound in the treatment of human colorectal cancer. We observed the in vivo inhibitory effect of Amex7 (1.25, 6.25, and 12.5 ml/kg, oral administration, twice daily) on tumor growth in a mouse model xenografted with HT-29 human colorectal cancer cells. In vitro, at 6, 12, and 24 h after 4% Amex7 treatment, we analyzed cell cycle by flow cytometry and the expression levels of cell cycle progression, apoptosis, and DNA damage repair-related proteins using immunoblotting and immunofluorescence staining in HT-29 cell line. As a result, Amex7 significantly suppressed tumor growth in HT-29 human colorectal cancer cells and xenografts. In vitro, Amex7 induced G2/M arrest through the regulation of cell cycle proteins and cell death by apoptosis and autophagy. Additionally, Amex7 consistently induced DNA damage and delayed the repair of Amex7-induced DNA damage by reducing the level of HR repair proteins. In conclusion, Amex7 enhanced anticancer effects through the induction of G2/M arrest and cell death, including apoptosis and autophagy. Furthermore, Amex7 impaired DNA damage repair. The present study provides a scientific rationale for the clinical use of a combined mixture of medicinal mushrooms and P. ginseng root extracts as an adjuvant treatment in human colorectal cancer.

Engineered Mesenchymal Cells Improve Passive Immune Protection Against Lethal Venezuelan Equine Encephalitis Virus Exposure

PubMed Central

Braid, Lorena R.; Davies, John E.; Nagata, Les P.

2016-01-01

Mesenchymal stromal cells (MSCs) are being exploited as gene delivery vectors for various disease and injury therapies. We provide proof-of-concept that engineered MSCs can provide a useful, effective platform for protection against infectious disease. Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne pathogen affecting humans and equines and can be used in bio-warfare. No licensed vaccine or antiviral agent currently exists to combat VEEV infection in humans. Direct antibody administration (passive immunity) is an effective, but short-lived, method of providing immediate protection against a pathogen. We compared the protective efficacy of human umbilical cord perivascular cells (HUCPVCs; a rich source of MSCs), engineered with a transgene encoding a humanized VEEV-neutralizing antibody (anti-VEEV), to the purified antibody. In athymic mice, the anti-VEEV antibody had a half-life of 3.7 days, limiting protection to 2 or 3 days after administration. In contrast, engineered HUCPVCs generated protective anti-VEEV serum titers for 21–38 days after a single intramuscular injection. At 109 days after transplantation, 10% of the mice still had circulating anti-VEEV antibody. The mice were protected against exposure to a lethal dose of VEEV by an intramuscular pretreatment injection with engineered HUCPVCs 24 hours or 10 days before exposure, demonstrating both rapid and prolonged immune protection. The present study is the first to describe engineered MSCs as gene delivery vehicles for passive immunity and supports their utility as antibody delivery vehicles for improved, single-dose prophylaxis against endemic and intentionally disseminated pathogens. Significance Direct injection of monoclonal antibodies (mAbs) is an important strategy to immediately protect the recipient from a pathogen. This strategy is critical during natural outbreaks or after the intentional release of bio-weapons. Vaccines require weeks to become effective, which is not practical for first responders immediately deployed to an infected region. However, mAb recipients often require booster shots to maintain protection, which is expensive and impractical once the first responders have been deployed. The present study has shown, for the first time, that mesenchymal stromal cells are effective gene delivery vehicles that can significantly improve mAb-mediated immune protection in a single, intramuscular dose of engineered cells. Such a cell-based delivery system can provide extended life-saving protection in the event of exposure to biological threats using a more practical, single-dose regimen. PMID:27334491

Suppression of intestinal carcinogenesis in Apc-mutant mice by limonin.

PubMed

Shimizu, Satomi; Miyamoto, Shingo; Fujii, Gen; Nakanishi, Ruri; Onuma, Wakana; Ozaki, Yoshihiko; Fujimoto, Kyoko; Yano, Tomohiro; Mutoh, Michihiro

2015-07-01

Limonoids in citrus fruits are known to possess multiple biological functions, such as anti-proliferative functions in human cancer cell lines. Therefore, we aimed to investigate the suppressive effect of limonin on intestinal polyp development in Apc-mutant Min mice. Five-week-old female Min mice were fed a basal diet or a diet containing 250 or 500 ppm limonin for 8 weeks. The total number of polyps in mice treated with 500 ppm limonin decreased to 74% of the untreated control value. Neoplastic cell proliferation in the polyp parts was assessed by counting PCNA positive cells, and a tendency of reduction was obtained by limonin treatment. Moreover, expression levels of c-Myc and MCP-1 mRNA in the polyp part were reduced by administration of limonin. We finally confirmed the effects of limonin on β-catenin signaling, and found limonin significantly inhibited T-cell factor/lymphocyte enhancer factor-dependent transcriptional activity in a dose-dependent manner in the Caco-2 human colon cancer cell line. Our results suggest that limonin might be a candidate chemopreventive agent against intestinal carcinogenesis.

Chiral effects in adrenocorticolytic action of o,p'-DDD (mitotane) in human adrenal cells.

PubMed

Asp, V; Cantillana, T; Bergman, A; Brandt, I

2010-03-01

Adrenocortical carcinoma (ACC) is a rare malignant disease with poor prognosis. The main pharmacological choice, o,p'-DDD (mitotane), produces severe adverse effects. Since o,p'-DDD is a chiral molecule and stereoisomers frequently possess different pharmacokinetic and/or pharmacodynamic properties, we isolated the two o,p'-DDD enantiomers, (R)-(+)-o,p'-DDD and (S)-(-)-o,p'-DDD, and determined their absolute structures. The effects of each enantiomer on cell viability and on cortisol and dehydroepiandrosterone (DHEA) secretion in the human adrenocortical cell line H295R were assessed. We also assayed the o,p'-DDD racemate and the m,p'- and p,p'-isomers. The results show small but statistically significant differences in activity of the o,p'-DDD enantiomers for all parameters tested. The three DDD isomers were equally potent in decreasing cell viability, but p,p'-DDD affected hormone secretion slightly less than the o,p'- and m,p'-isomers. The small chiral differences in direct effects on target cells alone do not warrant single enantiomer administration, but might reach importance in conjunction with possible stereochemical effects on pharmacokinetic processes in vivo.

Clitocine potentiates TRAIL-mediated apoptosis in human colon cancer cells by promoting Mcl-1 degradation.

PubMed

Sun, Jian-Guo; Ruan, Feng; Zeng, Xue-Li; Xiang, Jun; Li, Xia; Wu, Ping; Fung, Kwok Pui; Liu, Fei-Yan

2016-10-01

Among anti-cancer candidate drugs, TRAIL might be the most specific agent against cancer cells due to its low toxicity to normal cells. Unfortunately, cancer cells usually develop drug resistance to TRAIL, which is a major obstacle for its clinical application. One promising strategy is co-administrating with sensitizer to overcome cancer cells resistance to TRAIL. Clitocine, a natural amino nucleoside purified from wild mushroom, is recently demonstrated that can induce apoptosis in multidrug-resistant human cancer cells by targeting Mcl-1. In the present study,we found that pretreatment with clitocine dramatically enhances TRAIL lethality in its resistant human colon cancer cells by inducing apoptosis. More importantly, combination of clitocine and TRAIL also effectively inhibits xenograft growth and induces tumor cells apoptosis in athymic mice. The disruption of the binding between Mcl-1 and Bak as well as mitochondrial translocation of Bax mediated by clitocine are identified as the key underlying mechanisms, which leading to mitochondrial membrane permeabilization. Enforced exogenous Mcl-1 can effectively attenuate clitocine/TRAIL-induced apoptosis by suppressing the activation of intrinsic apoptotic pathway. Furthermore, clitocine regulates Mcl-1 expression at the posttranslational level as no obvious change is observed on mRNA level and proteasome inhibitor MG132 almost blocks the Mcl-1 suppression by clitocine. In fact, more ubiquitinated Mcl-1 was detected under clitocine treatment. Our findings indicate that clitocine is potentially an effective adjuvant agent in TRAIL-based cancer therapy.

Short-term administration of rhGH increases markers of cellular proliferation but not milk protein gene expression in normal lactating women

PubMed Central

Maningat, Patricia D.; Sen, Partha; Rijnkels, Monique; Hadsell, Darryl L.; Bray, Molly S.

2011-01-01

Growth hormone is one of few pharmacologic agents known to augment milk production in humans. We hypothesized that recombinant human GH (rhGH) increases the expression of cell proliferation and milk protein synthesis genes. Sequential milk and blood samples collected over four days were obtained from five normal lactating women. Following 24 h of baseline milk and blood sampling, rhGH (0.1 mg/kg/day) was administered subcutaneously once daily for 3 days. Gene expression changes were determined by microarray studies utilizing milk fat globule RNA isolated from each milk sample. Following rhGH administration, DNA synthesis and cell cycle genes were induced, while no significant changes were observed in the expression of milk synthesis genes. Expression of glycolysis and citric acid cycle genes were increased by day 4 compared with day 1, while lipid synthesis genes displayed a circadian-like pattern. Cell cycle gene upregulation occurred after a lag of ∼2 days, likely explaining the failure to increase milk production after only 3 days of rhGH treatment. We conclude that rhGH induces expression of cellular proliferation and metabolism genes but does not induce milk protein gene expression, as potential mechanisms for increasing milk production and could account for the known effect of rhGH to increase milk production following 7–10 days. PMID:21205870

Genetic modification of neurons to express bevacizumab for local anti-angiogenesis treatment of glioblastoma.

PubMed

Hicks, Martin J; Funato, Kosuke; Wang, Lan; Aronowitz, Eric; Dyke, Jonathan P; Ballon, Douglas J; Havlicek, David F; Frenk, Esther Z; De, Bishnu P; Chiuchiolo, Maria J; Sondhi, Dolan; Hackett, Neil R; Kaminsky, Stephen M; Tabar, Viviane; Crystal, Ronald G

2015-01-01

The median survival of glioblastoma multiforme (GBM) is approximately 1 year. Following surgical removal, systemic therapies are limited by the blood-brain barrier. To circumvent this, we developed a method to modify neurons with the genetic sequence for therapeutic monoclonal antibodies using adeno-associated virus (AAV) gene transfer vectors, directing persistent, local expression in the tumor milieu. The human U87MG GBM cell line or patient-derived early passage GBM cells were administered to the striatum of NOD/SCID immunodeficient mice. AAVrh.10BevMab, an AAVrh.10-based vector coding for bevacizumab (Avastin), an anti-human vascular endothelial growth factor (VEGF) monoclonal antibody, was delivered to the area of the GBM xenograft. Localized expression of bevacizumab was demonstrated by quantitative PCR, ELISA and western blotting. Immunohistochemistry showed that bevacizumab was expressed in neurons. Concurrent administration of AAVrh.10BevMab with the U87MG tumor reduced tumor blood vessel density and tumor volume, and increased survival. Administration of AAVrh.10BevMab 1 week after U87MG xenograft reduced growth and increased survival. Studies with patient-derived early passage GBM primary cells showed a reduction in primary tumor burden with an increased survival. These data support the strategy of AAV-mediated central nervous system gene therapy to treat GBM, overcoming the blood-brain barrier through local, persistent delivery of an anti-angiogenesis monoclonal antibody.

Immunomodulating and anti-allergic effects of Negroamaro and Koshu Vitis vinifera fermented grape marc (FGM).

PubMed

Marzulli, Giuseppe; Magrone, Thea; Vonghia, Luisa; Kaneko, Masahiro; Takimoto, Hiroaki; Kumazawa, Yoshio; Jirillo, Emilio

2014-01-01

Polyphenols contained in FGM from Negroamaro (N) and Koshu (K) Vitis vinifera have been shown to exhibit several immunomodulating activities. For instance, mice affected by experimental colitis when administered with K-FGM showed an attenuation of the inflammatory process. In murine asthma, K-FGM reduced IgE production and eosinophil number in bronchial alveolar lavage fluid. In vitro, both N- and K-FGM were able to induce T regulatory cells in terms of Foxp-3 molecule expression and release of interleukin-10. In another set of experiments both N- and K-FGM were able to balance rate of proliferation/apoptosis/necrosis of normal human peripheral lymphocytes, thus indicating the property of these compounds to maintain immune homeostatic mechanisms in the host. On the other hand, N- and K-FGM inhibited human basophil degranulation, thus, confirming our previous results obtained with rat basophilic leukemia cells. Finally, N- and K-FGM also decreased oxidative burst of human polymorphonuclear cells and monocytes.Taken together, these findings imply the potential clinical usefulness of FGM administration in inflammatory/allergic conditions, such as chronic asthma.

TOPK inhibitor induces complete tumor regression in xenograft models of human cancer through inhibition of cytokinesis.

PubMed

Matsuo, Yo; Park, Jae-Hyun; Miyamoto, Takashi; Yamamoto, Shinji; Hisada, Shoji; Alachkar, Houda; Nakamura, Yusuke

2014-10-22

TOPK (T-lymphokine-activated killer cell-originated protein kinase) is highly and frequently transactivated in various cancer tissues, including lung and triple-negative breast cancers, and plays an indispensable role in the mitosis of cancer cells. We report the development of a potent TOPK inhibitor, OTS964 {(R)-9-(4-(1-(dimethylamino)propan-2-yl)phenyl)-8-hydroxy-6-methylthieno[2,3-c]quinolin-4(5H)-one}, which inhibits TOPK kinase activity with high affinity and selectivity. Similar to the knockdown effect of TOPK small interfering RNAs (siRNAs), this inhibitor causes a cytokinesis defect and the subsequent apoptosis of cancer cells in vitro as well as in xenograft models of human lung cancer. Although administration of the free compound induced hematopoietic adverse reactions (leukocytopenia associated with thrombocytosis), the drug delivered in a liposomal formulation effectively caused complete regression of transplanted tumors without showing any adverse reactions in mice. Our results suggest that the inhibition of TOPK activity may be a viable therapeutic option for the treatment of various human cancers. Copyright © 2014, American Association for the Advancement of Science.

45 CFR 1177.8 - Administrative offset.

Code of Federal Regulations, 2010 CFR

2010-10-01

... HUMANITIES NATIONAL ENDOWMENT FOR THE HUMANITIES CLAIMS COLLECTION § 1177.8 Administrative offset. (a) The... administrative offset, the National Endowment for the Humanities will send the debtor written notice of the... interest. (d) The National Endowment for the Humanities may effect an administrative offset against a...

45 CFR 1177.8 - Administrative offset.

Code of Federal Regulations, 2011 CFR

2011-10-01

... HUMANITIES NATIONAL ENDOWMENT FOR THE HUMANITIES CLAIMS COLLECTION § 1177.8 Administrative offset. (a) The... administrative offset, the National Endowment for the Humanities will send the debtor written notice of the... interest. (d) The National Endowment for the Humanities may effect an administrative offset against a...

Chemokine Ligand 20: A Signal for Leukocyte Recruitment During Human Ovulation?

PubMed

Al-Alem, Linah; Puttabyatappa, Muraly; Rosewell, Kathy; Brännström, Mats; Akin, James; Boldt, Jeffrey; Muse, Ken; Curry, Thomas E

2015-09-01

Ovulation is one of the cornerstones of female fertility. Disruption of the ovulatory process results in infertility, which affects approximately 10% of couples. Using a unique model in which the dominant follicle is collected across the periovulatory period in women, we have identified a leukocyte chemoattractant, chemokine ligand 20 (CCL20), in the human ovary. CCL20 mRNA is massively induced after an in vivo human chorionic gonadotropin (hCG) stimulus in granulosa (>10 000-fold) and theca (>4000-fold) cells collected during the early ovulatory (12-18 h) and late ovulatory (18-34 h) periods after hCG administration. Because the LH surge sets in motion an inflammatory reaction characterized by an influx of leukocytes and CCL20 is known to recruit leukocytes in other systems, the composition of ovarian leukocytes (CD45+) containing the CCL20 receptor CCR6 was determined immediately prior to ovulation. CD45+/CCR6+ cells were primarily natural killer cells (41%) along with B cells (12%), T cells (11%), neutrophils (10%), and monocytes (9%). Importantly, exogenous CCL20 stimulated ovarian leukocyte migration 59% within 90 minutes. Due to the difficulties in obtaining human follicles, an in vitro model was developed using granulosa-lutein cells to explore CCL20 regulation. CCL20 expression increased 40-fold within 6 hours after hCG, was regulated partially by the epithelial growth factor pathway, and was positively correlated with progesterone production. These results demonstrate that hCG dramatically increases CCL20 expression in the human ovary, that ovarian leukocytes contain the CCL20 receptor, and that CCL20 stimulates leukocyte migration. Our findings raise the prospect that CCL20 may aid in the final ovulatory events and contribute to fertility in women.

Chemokine Ligand 20: A Signal for Leukocyte Recruitment During Human Ovulation?

PubMed Central

Al-Alem, Linah; Puttabyatappa, Muraly; Rosewell, Kathy; Brännström, Mats; Akin, James; Boldt, Jeffrey; Muse, Ken

2015-01-01

Ovulation is one of the cornerstones of female fertility. Disruption of the ovulatory process results in infertility, which affects approximately 10% of couples. Using a unique model in which the dominant follicle is collected across the periovulatory period in women, we have identified a leukocyte chemoattractant, chemokine ligand 20 (CCL20), in the human ovary. CCL20 mRNA is massively induced after an in vivo human chorionic gonadotropin (hCG) stimulus in granulosa (>10 000-fold) and theca (>4000-fold) cells collected during the early ovulatory (12–18 h) and late ovulatory (18–34 h) periods after hCG administration. Because the LH surge sets in motion an inflammatory reaction characterized by an influx of leukocytes and CCL20 is known to recruit leukocytes in other systems, the composition of ovarian leukocytes (CD45+) containing the CCL20 receptor CCR6 was determined immediately prior to ovulation. CD45+/CCR6+ cells were primarily natural killer cells (41%) along with B cells (12%), T cells (11%), neutrophils (10%), and monocytes (9%). Importantly, exogenous CCL20 stimulated ovarian leukocyte migration 59% within 90 minutes. Due to the difficulties in obtaining human follicles, an in vitro model was developed using granulosa-lutein cells to explore CCL20 regulation. CCL20 expression increased 40-fold within 6 hours after hCG, was regulated partially by the epithelial growth factor pathway, and was positively correlated with progesterone production. These results demonstrate that hCG dramatically increases CCL20 expression in the human ovary, that ovarian leukocytes contain the CCL20 receptor, and that CCL20 stimulates leukocyte migration. Our findings raise the prospect that CCL20 may aid in the final ovulatory events and contribute to fertility in women. PMID:26125463