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author:("Das, hirano")
1.  Human Vγ2Vδ2 T cells limit breast cancer growth by modulating cell survival-, apoptosis-related molecules and microenvironment in tumors 
Innate immune system has been known to play an important role in inhibiting the malignant transformation, tumor progression and invasion. However, the mechanistic basis remains ambiguous. Despite polyclonality of human γδ T cells, Vγ2Vδ2 T cell subset was shown to recognize and limit the growth of various tumors at various degrees. The differential recognition of the tumor cells by Vγ2Vδ2 T cells are yet to be defined. Our study reveals that γδ T cells limit in vitro growth of most breast tumor cells, such as SkBr7 (HER2+), MCF7 (ER+) and MDA-MB-231 (ER−) by inhibiting their survival and inducing apoptosis, except BrCa-MZ01 (PR+) cells. To investigate detail mechanisms of antineoplastic effects, we found that cell death was associated with the surface expression levels of MICA/B and ICAM1. Molecular signaling analysis demonstrated that inhibition of cell growth by γδ T cells was associated with the lower expression levels of cell survival-related molecules such as AKT, ERK and concomitant upregulation of apoptosis-related molecules, such as PARP, cleaved caspase 3 and tumor suppressor genes PTEN and P53. However, opposite molecular signaling was observed in the resistant cell line after coculture with γδ T cells. In vivo, antineoplastic effects of γδ T cells were also documented, where tumor growth was inhibited due to the downregulation of survival signals, strong induction of apoptotic molecules, disruption of microvasculature and increased infiltration of tumor associated macrophages. These findings reveal that a complex molecular signaling is involved in γδ T cell-mediated antineoplastic effects.
doi:10.1002/ijc.28217
PMCID: PMC3939063  PMID: 23595559
Vγ2Vδ2 T cell; breast cancer; cell survival; apoptosis; angiogenesis; xenotransplant; NOD/SCID mice
2.  Correction: Impact of Diffusion Barriers to Small Cytotoxic Molecules on the Efficacy of Immunotherapy in Breast Cancer 
PLoS ONE  2013;8(10):10.1371/annotation/94eab1ba-657a-445d-abf2-a709fc6a9806.
doi:10.1371/annotation/94eab1ba-657a-445d-abf2-a709fc6a9806
PMCID: PMC3808440  PMID: 24204496
3.  Down-Regulation of the Oncogene PTTG1 via the KLF6 Tumor Suppressor during Induction of Myeloid Differentiation 
PLoS ONE  2013;8(8):e71282.
The aberrant expression of proto-oncogenes is involved in processes that are responsible for cellular proliferation and the inhibition of myeloid differentiation in acute myeloid leukemia (AML). Pituitary Tumor-Transforming gene 1 (PTTG1), an oncogenic transcription factor, is abundantly expressed in various human cancers and hematopoietic malignancies. However, its expression in normal leukocytes and most normal tissues is very low or undetectable. The mechanism by which PTTG1 overexpression modifies myeloid cell development and promotes leukemogenesis remain unclear. To investigate the mechanistic links between PTTG1 overexpression and leukemia cell differentiation, we utilized phorbol 12-myristate 13-acetate (PMA), a well-known agent that triggers monocyte/macrophage differentiation, to analyze the expression patterns of PTTG1 in PMA-induced myeloid differentiation. We found that PTTG1 is down-regulated at the transcriptional level in PMA-treated HL-60 and THP1 cells. In addition, we identified a binding site for a tumor suppressor protein, Kruppel-like factor 6 (KLF6), in the PTTG1 promoter. We found that KLF6 could directly bind and repress PTTG1 expression. In HL-60 and THP1 cells, KLF6 mRNA and protein levels are up-regulated with a concordant reduction of PTTG1 expression upon treatment with PMA. Furthermore, KLF6 knockdown by shRNA abolished the suppression of PTTG1 and reduced the activation of the differentiation marker CD11b in PMA-primed cells. The protein kinase C (PKC) inhibitor and the MAPK/ERK kinase (MEK) inhibitor significantly blocked the potentiation of PMA-mediated KLF6 induction and the down-regulation of PTTG1, indicating that PTTG1 is suppressed via the activation of PKC/ERK/KLF6 pathway. Our findings suggest that drugs that increase the KLF6 inhibition of PTTG1 may have a therapeutic application in AML treatment strategies.
doi:10.1371/journal.pone.0071282
PMCID: PMC3745464  PMID: 23977008
4.  Partially Silencing Brain Toll-Like Receptor 4 Prevents in Part Left Ventricular Remodeling with Sympathoinhibition in Rats with Myocardial Infarction-Induced Heart Failure 
PLoS ONE  2013;8(7):e69053.
Background
Left ventricular (LV) remodeling and activation of sympathetic nervous system (SNS) are cardinal features of heart failure. We previously demonstrated that enhanced central sympathetic outflow is associated with brain toll-like receptor 4 (TLR4) probably mediated by brain angiotensin II type 1 receptor in mice with myocardial infarction (MI)-induced heart failure. The purpose of the present study was to examine whether silencing brain TLR4 could prevent LV remodeling with sympathoinhibition in MI-induced heart failure.
Methodology/Principal Findings
MI-induced heart failure model rats were created by ligation of left coronary artery. The expression level of TLR4 in brainstem was significantly higher in MI-induced heart failure treated with intracerebroventricular (ICV) injection of hGAPDH-SiRNA than in sham. TLR4 in brainstem was significantly lower in MI-induced heart failure treated with ICV injection of TLR4-SiRNA than in that treated with ICV injection of hGAPDH-SiRNA. Lung weight, urinary norepinephrine excretion, and LV end-diastolic pressure were significantly lower and LV dimension was significantly smaller in MI-induced heart failure treated with TLR4-SiRNA than in that treated with hGAPDH-SiRNA for 2 weeks.
Conclusions
Partially silencing brain TLR4 by ICV injection of TLR4-SiRNA for 2 weeks could in part prevent LV remodeling with sympathoinhibition in rats with MI-induced heart failure. Brain TLR4 has a potential to be a target of the treatment for MI-induced heart failure.
doi:10.1371/journal.pone.0069053
PMCID: PMC3706428  PMID: 23874864
5.  Impact of Diffusion Barriers to Small Cytotoxic Molecules on the Efficacy of Immunotherapy in Breast Cancer 
PLoS ONE  2013;8(4):e61398.
Molecular-focused cancer therapies, e.g., molecularly targeted therapy and immunotherapy, so far demonstrate only limited efficacy in cancer patients. We hypothesize that underestimating the role of biophysical factors that impact the delivery of drugs or cytotoxic cells to the target sites (for associated preferential cytotoxicity or cell signaling modulation) may be responsible for the poor clinical outcome. Therefore, instead of focusing exclusively on the investigation of molecular mechanisms in cancer cells, convection-diffusion of cytotoxic molecules and migration of cancer-killing cells within tumor tissue should be taken into account to improve therapeutic effectiveness. To test this hypothesis, we have developed a mathematical model of the interstitial diffusion and uptake of small cytotoxic molecules secreted by T-cells, which is capable of predicting breast cancer growth inhibition as measured both in vitro and in vivo. Our analysis shows that diffusion barriers of cytotoxic molecules conspire with γδ T-cell scarcity in tissue to limit the inhibitory effects of γδ T-cells on cancer cells. This may increase the necessary ratios of γδ T-cells to cancer cells within tissue to unrealistic values for having an intended therapeutic effect, and decrease the effectiveness of the immunotherapeutic treatment.
doi:10.1371/journal.pone.0061398
PMCID: PMC3631240  PMID: 23620747
6.  IKKi Deficiency Promotes Pressure Overload-Induced Cardiac Hypertrophy and Fibrosis 
PLoS ONE  2013;8(1):e53412.
The inducible IκB kinase (IKKi/IKKε) is a recently described serine-threonine IKK-related kinase. Previous studies have reported the role of IKKi in infectious diseases and cancer. However, its role in the cardiac response to pressure overload remains elusive. In this study, we investigated the effects of IKKi deficiency on the development of pathological cardiac hypertrophy using in vitro and in vivo models. First, we developed mouse models of pressure overload cardiac hypertrophy induced by pressure overload using aortic banding (AB). Four weeks after AB, cardiac function was then assessed through echocardiographic and hemodynamic measurements. Western blotting, real-time PCR and histological analyses were used to assess the pathological and molecular mechanisms. We observed that IKKi-deficient mice showed significantly enhanced cardiac hypertrophy, cardiac dysfunction, apoptosis and fibrosis compared with WT mice. Furthermore, we recently revealed that the IKKi-deficient mice spontaneously develop cardiac hypertrophy. Moreover, in vivo experiments showed that IKKi deficiency-induced cardiac hypertrophy was associated with the activation of the AKT and NF-κB signaling pathway in response to AB. In cultured cells, IKKi overexpression suppressed the activation of this pathway. In conclusion, we demonstrate that IKKi deficiency exacerbates cardiac hypertrophy by regulating the AKT and NF-κB signaling pathway.
doi:10.1371/journal.pone.0053412
PMCID: PMC3551922  PMID: 23349709
7.  Human Umbilical Cord Blood-Derived CD34+ Cells Reverse Osteoporosis in NOD/SCID Mice by Altering Osteoblastic and Osteoclastic Activities 
PLoS ONE  2012;7(6):e39365.
Background
Osteoporosis is a bone disorder associated with loss of bone mineral density and micro architecture. A balance of osteoblasts and osteoclasts activities maintains bone homeostasis. Increased bone loss due to increased osteoclast and decreased osteoblast activities is considered as an underlying cause of osteoporosis.
Methods and Findings
The cures for osteoporosis are limited, consequently the potential of CD34+ cell therapies is currently being considered. We developed a nanofiber-based expansion technology to obtain adequate numbers of CD34+ cells isolated from human umbilical cord blood, for therapeutic applications. Herein, we show that CD34+ cells could be differentiated into osteoblastic lineage, in vitro. Systemically delivered CD34+ cells home to the bone marrow and significantly improve bone deposition, bone mineral density and bone micro-architecture in osteoporotic mice. The elevated levels of osteocalcin, IL-10, GM-CSF, and decreased levels of MCP-1 in serum parallel the improvements in bone micro-architecture. Furthermore, CD34+ cells improved osteoblast activity and concurrently impaired osteoclast differentiation, maturation and functionality.
Conclusions
These findings demonstrate a novel approach utilizing nanofiber-expanded CD34+ cells as a therapeutic application for the treatment of osteoporosis.
doi:10.1371/journal.pone.0039365
PMCID: PMC3377665  PMID: 22724005
8.  Age and Ovariectomy Abolish Beneficial Effects of Female Sex on Rat Ventricular Myocytes Exposed to Simulated Ischemia and Reperfusion 
PLoS ONE  2012;7(6):e38425.
Sex differences in responses to myocardial ischemia have been described, but whether cardiomyocyte function is influenced by sex in the setting of ischemia and reperfusion has not been elucidated. This study compared contractions and intracellular Ca2+ in isolated ventricular myocytes exposed to ischemia and reperfusion. Cells were isolated from anesthetized 3-month-old male and female Fischer 344 rats, paced at 4 Hz (37°C), exposed to simulated ischemia (20 mins) and reperfused. Cell shortening (edge detector) and intracellular Ca2+ (fura-2) were measured simultaneously. Cell viability was assessed with Trypan blue. Ischemia reduced peak contractions and increased Ca2+ levels equally in myocytes from both sexes. However, contraction amplitudes were reduced in reperfusion in male myocytes, while contractions recovered to exceed control levels in females (62.6±5.1 vs. 140.1±15.8%; p<0.05). Only 60% of male myocytes excluded trypan blue dye after ischemia and reperfusion, while all female cardiomyocytes excluded the dye (p<0.05). Parallel experiments were conducted in myocytes from ∼24-month-old female rats or 5–6-month-old rats that had an ovariectomy at 3–4 weeks of age. Beneficial effects of female sex on myocyte viability and contractile dysfunction in reperfusion were abolished in cells from 24-month-old females. Aged female myocytes also exhibited elevated intracellular Ca2+ and alternans in ischemia. Cells from ovariectomized rats displayed increased Ca2+ transients and spontaneous activity in ischemia compared to sham-operated controls. None of the myocytes from ovariectomized rats were viable after 15 minutes of ischemia, while 75% of sham cells remained viable at end of reperfusion (p<0.05). These findings demonstrate that cardiomyocytes from young adult females are more resistant to ischemia and reperfusion injury than cells from males. Age and OVX abolish these beneficial effects and induce Ca2+ dysregulation at the level of the cardiomyocyte. Thus, beneficial effects of estrogen in ischemia and reperfusion are mediated, in part, by effects on cardiomyocytes.
doi:10.1371/journal.pone.0038425
PMCID: PMC3368849  PMID: 22701638
9.  Effects of PPARs Agonists on Cardiac Metabolism in Littermate and Cardiomyocyte-Specific PPAR-γ –Knockout (CM-PGKO) Mice 
PLoS ONE  2012;7(4):e35999.
Understanding the molecular regulatory mechanisms controlling for myocardial lipid metabolism is of critical importance for the development of new therapeutic strategies for heart diseases. The role of PPARγ and thiazolidinediones in regulation of myocardial lipid metabolism is controversial. The aim of our study was to assess the role of PPARγ on myocardial lipid metabolism and function and differentiate local/from systemic actions of PPARs agonists using cardiomyocyte-specific PPARγ –knockout (CM-PGKO) mice. To this aim, the effect of PPARγ, PPARγ/PPARα and PPARα agonists on cardiac function, intra-myocyte lipid accumulation and myocardial expression profile of genes and proteins, affecting lipid oxidation, uptake, synthesis, and storage (CD36, CPT1MIIA, AOX, FAS, SREBP1-c and ADPR) was evaluated in cardiomyocyte-specific PPARγ –knockout (CM-PGKO) and littermate control mice undergoing standard and high fat diet (HFD). At baseline, protein levels and mRNA expression of genes involved in lipid uptake, oxidation, synthesis, and accumulation of CM-PGKO mice were not significantly different from those of their littermate controls. At baseline, no difference in myocardial lipid content was found between CM-PGKO and littermate controls. In standard condition, pioglitazone and rosiglitazone do not affect myocardial metabolism while, fenofibrate treatment significantly increased CD36 and CPT1MIIA gene expression. In both CM-PGKO and control mice submitted to HFD, six weeks of treatment with rosiglitazone, fenofibrate and pioglitazone lowered myocardial lipid accumulation shifting myocardial substrate utilization towards greater contribution of glucose. In conclusion, at baseline, PPARγ does not play a crucial role in regulating cardiac metabolism in mice, probably due to its low myocardial expression. PPARs agonists, indirectly protect myocardium from lipotoxic damage likely reducing fatty acids delivery to the heart through the actions on adipose tissue. Nevertheless a direct non- PPARγ mediated mechanism of PPARγ agonist could not be ruled out.
doi:10.1371/journal.pone.0035999
PMCID: PMC3338561  PMID: 22563432
10.  Embryonic Stem Cell-Derived Factors Inhibit T Effector Activation and Induce T Regulatory Cells by Suppressing PKC-θ Activation 
PLoS ONE  2012;7(3):e32420.
Embryonic stem cells (ESCs) possess immune privileged properties and have the capacity to modulate immune activation. However, the mechanisms by which ESCs inhibit immune activation remain mostly unknown. We have previously shown that ESC-derived factors block dendritic cell maturation, thereby indirectly affecting T cell activation. Here, we show that ESC-derived factors also directly affect T cell activation. We provide the first demonstration that ESC-derived factors significantly down-regulated the expressions of IL-2 and IFN-γ, while markedly up-regulating the expression of IL-10, TGF-β, and Treg transcription factor Foxp3 in CD4+ CD25+ T cells. Furthermore, ESC-derived factors robustly suppressed T cell proliferation in response to the protein kinase C-θ (PKC-θ) activator phorbol 12-myristate 13-acetate (PMA). Western blot analysis indicated that ESC-derived factors prevented PKC-θ phosphorylation without influencing total PKC-θ levels. Moreover, IκB-α degradation was abrogated, confirming absence of PKC-θ activity. The impact of ESC-derived factors on PKC-θ activation appeared to be specific since other upstream T cell signaling components were not affected. In conclusion, ESCs appear to directly impact T cell activation and polarization by negatively regulating the PKC-θ pathway.
doi:10.1371/journal.pone.0032420
PMCID: PMC3296708  PMID: 22412871
11.  Plasticity and Maintenance of Hematopoietic Stem Cells During Development 
Maintenance of hematopoietic stem cells (HSCs) pool depends on fine balance between self-renewal and differentiation of HSCs. HSCs normally reside within the bone marrow niche of an adult mammal. The embryonic development of HSCs is a complex process that involves the migration of developing HSCs in multiple anatomical sites. Throughout the process, developing HSCs receive internal (transcriptional program) and external (HSC niche) signals, which direct them to maintain balance between self-renewal and differentiation, also to generate a pool of HSCs. In physiological condition HSCs differentiate into all mature cell types present in the blood. However, in pathological condition they may differentiate into non-hematological cells according to the need of the body. It was shown that HSCs can transdifferentiate into cell types that do not belong to the hematopoietic system suggests a complete paradigm shift of the hierarchical hematopoietic tree. This review describes the developmental origins and regulation of HSCs focusing on developmental signals that induce the adult hematopoietic stem cell program, as these informations are very critical for manipulating conditions for expansion of HSCs in ex vivo condition. This review also states clinical application and related patents using HSC.
PMCID: PMC3294454  PMID: 21517745
Hematopoietic stem cells; embryonic development; regulation; self-renewal; differentiation; transdifferentiation; clinical application; patents
12.  Safety and efficacy of bone marrow-derived autologous CD133+ stem cell therapy 
The Phase I clinical study was designed to assess the safety and feasibility of a dose escalating intracoronary infusion of autologous bone marrow (BM)-derived CD133+ stem cell therapy to the patients with chronic total occlusion (CTO) and ischemia. Nine patients were received CD133+ cells into epicardial vessels supplying collateral flow to areas of viable ischemic myocardium in the distribution of the CTO. There were no major adverse cardiac events (MACE), revascularization, re-admission to the hospital secondary to angina, or acute myocardial infarction (AMI) for the 24-month period following cellular infusion. In addition, there were no periprocedural infusion-related complications including malignant arrhythmias, loss of normal coronary blood flow or acute neurologic events. Cardiac enzymes were negative in all patients. There was an improvement in the degree of ischemic myocardium, which was accompanied by a trend towards reduction in anginal symptoms. Intracoronary infusion of autologous CD133+ marrow-derived cells is safe and feasible. Cellular therapy with CD133+ cells to reduce anginal symptoms and to improve ischemia in patients with CTO awaits clinical investigation in Phase II/III trials.
PMCID: PMC3285247  PMID: 21196330
Safety; Efficacy; Autologous; Bone marrow; CD133; Chronic; Myocardial; Ischemia; Therapy
13.  Pharmacological Inhibition of Caspase and Calpain Proteases: A Novel Strategy to Enhance the Homing Responses of Cord Blood HSPCs during Expansion 
PLoS ONE  2012;7(1):e29383.
Background
Expansion of hematopoietic stem/progenitor cells (HSPCs) is a well-known strategy employed to facilitate the transplantation outcome. We have previously shown that the prevention of apoptosis by the inhibition of cysteine proteases, caspase and calpain played an important role in the expansion and engraftment of cord blood (CB) derived HSPCs. We hypothesize that these protease inhibitors might have maneuvered the adhesive and migratory properties of the cells rendering them to be retained in the bone marrow for sustained engraftment. The current study was aimed to investigate the mechanism of the homing responses of CB cells during expansion.
Methodology/Principal Findings
CB derived CD34+ cells were expanded using a combination of growth factors with and without Caspase inhibitor -zVADfmk or Calpain 1 inhibitor- zLLYfmk. The cells were analyzed for the expression of homing-related molecules. In vitro adhesive/migratory interactions and actin polymerization dynamics of HSPCs were assessed. In vivo homing assays were carried out in NOD/SCID mice to corroborate these observations. We observed that the presence of zVADfmk or zLLYfmk (inhibitors) caused the functional up regulation of CXCR4, integrins, and adhesion molecules, reflecting in a higher migration and adhesive interactions in vitro. The enhanced actin polymerization and the RhoGTPase protein expression complemented these observations. Furthermore, in vivo experiments showed a significantly enhanced homing to the bone marrow of NOD/SCID mice.
Conclusion/Significance
Our present study reveals another novel aspect of the regulation of caspase and calpain proteases in the biology of HSPCs. The priming of the homing responses of the inhibitor-cultured HSPCs compared to the cytokine-graft suggests that the modulation of these proteases may help in overcoming the major homing defects prevalent in the expansion cultures thereby facilitating the manipulation of cells for transplant procedures.
doi:10.1371/journal.pone.0029383
PMCID: PMC3250442  PMID: 22235291
14.  Stepwise Maturation of Lytic Granules during Differentiation and Activation of Human CD8+ T Lymphocytes 
PLoS ONE  2011;6(11):e27057.
During differentiation, cytotoxic T lymphocytes (CTL) acquire their killing potential through the biogenesis and maturation of lytic granules that are secreted upon target cell recognition. How lytic granule load in lytic molecules evolves during CTL differentiation and which subsets of lytic granules are secreted following activation remains to be investigated. We set up a flow cytometry approach to analyze single lytic granules isolated from primary human CTL according to their size and molecular content. During CTL in vitro differentiation, a relatively homogeneous population of lytic granules appeared through the progressive loading of Granzyme B, Perforin and Granzyme A within LAMP1+ lysosomes. PMA/ionomycin-induced lytic granule exocytosis was preceded by a rapid association of the docking molecule Rab27a to approximately half of the lytic granules. Activated CTL were found to limit exocytosis by sparing lytic granules including some associated to Rab27a. Our study provides a quantification of key steps of lytic granule biogenesis and highlights the potential of flow cytometry to study organelle composition and dynamics.
doi:10.1371/journal.pone.0027057
PMCID: PMC3208563  PMID: 22073254
15.  Thymosin Beta 4 Prevents Oxidative Stress by Targeting Antioxidant and Anti-Apoptotic Genes in Cardiac Fibroblasts 
PLoS ONE  2011;6(10):e26912.
Rationale
Thymosin beta-4 (Tβ4) is a ubiquitous protein with diverse functions relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory responses. The effecter molecules targeted by Tβ4 for cardiac protection remains unknown. The purpose of this study is to determine the molecules targeted by Tβ4 that mediate cardio-protection under oxidative stress.
Methods
Rat neonatal fibroblasts cells were exposed to hydrogen peroxide (H2O2) in presence and absence of Tβ4 and expression of antioxidant, apoptotic and pro-fibrotic genes was evaluated by quantitative real-time PCR and western blotting. Reactive oxygen species (ROS) levels were estimated by DCF-DA using fluorescent microscopy and fluorimetry. Selected antioxidant and antiapoptotic genes were silenced by siRNA transfections in cardiac fibroblasts and the effect of Tβ4 on H2O2-induced profibrotic events was evaluated.
Results
Pre-treatment with Tβ4 resulted in reduction of the intracellular ROS levels induced by H2O2 in the cardiac fibroblasts. This was associated with an increased expression of antioxidant enzymes Cu/Zn superoxide dismutase (SOD) and catalase and reduction of Bax/Bcl2 ratio. Tβ4 treatment reduced the expression of pro-fibrotic genes [connective tissue growth factor (CTGF), collagen type-1 (Col-I) and collagen type-3 (Col-III)] in the cardiac fibroblasts. Silencing of Cu/Zn-SOD and catalase gene triggered apoptotic cell death in the cardiac fibroblasts, which was prevented by treatment with Tβ4.
Conclusion
This is the first report that exhibits the targeted molecules modulated by Tβ4 under oxidative stress utilizing the cardiac fibroblasts. Tβ4 treatment prevented the profibrotic gene expression in the in vitro settings. Our findings indicate that Tβ4 selectively targets and upregulates catalase, Cu/Zn-SOD and Bcl2, thereby, preventing H2O2-induced profibrotic changes in the myocardium. Further studies are warranted to elucidate the signaling pathways involved in the cardio-protection afforded by Tβ4.
doi:10.1371/journal.pone.0026912
PMCID: PMC3201979  PMID: 22046407
16.  Human Ovarian Tumor Cells Escape γδ T Cell Recognition Partly by Down Regulating Surface Expression of MICA and Limiting Cell Cycle Related Molecules 
PLoS ONE  2011;6(9):e23348.
Background
Mechanisms of human Vγ2Vδ2 T cell-mediated tumor immunity have yet to be fully elucidated.
Methods and Findings
At least some tumor cell recognition is mediated by NKG2D-MICA interactions. Herein, by using MTT assay and PI-BrdU co-staining and Western-blot, we show that these Vγ2Vδ2 T cells can limit the proliferation of ovarian tumor cells by down regulation of apoptosis and cell cycle related molecules in tumor cells. Cell-to-cell contact is critical. γδ T cell-resistant, but not susceptible ovarian tumor cells escape γδ T cell-mediated immune recognition by up-regulating pErk1/2, thereby decreasing surface MICA levels. Erk1/2 inhibitor pretreatment or incubation prevents this MICA decrease, while up-regulating key cell cycle related molecules such as CDK2, CDK4 and Cyclin D1, as well as apoptosis related molecules making resistant tumor cells now vulnerable to γδ T cell-mediated lysis.
Conclusion
These findings demonstrate novel effects of γδT cells on ovarian tumor cells.
doi:10.1371/journal.pone.0023348
PMCID: PMC3173356  PMID: 21935360
17.  Murine and Bovine γδ T Cells Enhance Innate Immunity against Brucella abortus Infections 
PLoS ONE  2011;6(7):e21978.
γδ T cells have been postulated to act as a first line of defense against infectious agents, particularly intracellular pathogens, representing an important link between the innate and adaptive immune responses. Human γδ T cells expand in the blood of brucellosis patients and are active against Brucella in vitro. However, the role of γδ T cells in vivo during experimental brucellosis has not been studied. Here we report TCRδ−/− mice are more susceptible to B. abortus infection than C57BL/6 mice at one week post-infection as measured by splenic colonization and splenomegaly. An increase in TCRγδ cells was observed in the spleens of B. abortus-infected C57BL/6 mice, which peaked at two weeks post-infection and occurred concomitantly with diminished brucellae. γδ T cells were the major source of IL-17 following infection and also produced IFN-γ. Depletion of γδ T cells from C57BL/6, IL-17Rα−/−, and GMCSF−/− mice enhanced susceptibility to B. abortus infection although this susceptibility was unaltered in the mutant mice; however, when γδ T cells were depleted from IFN-γ−/− mice, enhanced susceptibility was observed. Neutralization of γδ T cells in the absence of TNF-α did not further impair immunity. In the absence of TNF-α or γδ T cells, B. abortus-infected mice showed enhanced IFN-γ, suggesting that they augmented production to compensate for the loss of γδ T cells and/or TNF-α. While the protective role of γδ T cells was TNF-α-dependent, γδ T cells were not the major source of TNF-α and activation of γδ T cells following B. abortus infection was TNF-α-independent. Additionally, bovine TCRγδ cells were found to respond rapidly to B. abortus infection upon co-culture with autologous macrophages and could impair the intramacrophage replication of B. abortus via IFN-γ. Collectively, these results demonstrate γδ T cells are important for early protection to B. abortus infections.
doi:10.1371/journal.pone.0021978
PMCID: PMC3134454  PMID: 21765931
18.  High-Dose Chemotherapy Followed by Autologous Stem Cell Transplantation for Metastatic Rhabdomyosarcoma—A Systematic Review 
PLoS ONE  2011;6(2):e17127.
Introduction
Patients with metastatic rhabdomyosarcoma (RMS) have a poor prognosis. The aim of this systematic review is to investigate whether high-dose chemotherapy (HDCT) followed by autologous hematopoietic stem cell transplantation (HSCT) in patients with metastatic RMS has additional benefit or harm compared to standard chemotherapy.
Methods
Systematic literature searches were performed in MEDLINE, EMBASE, and The Cochrane Library. All databases were searched from inception to February 2010. PubMed was searched in June 2010 for a last update. In addition to randomized and non-randomized controlled trials, case series and case reports were included to complement results from scant data. The primary outcome was overall survival. A meta-analysis was performed using the hazard ratio as primary effect measure, which was estimated from Cox proportional hazard models or from summary statistics of Kaplan Meier product-limit estimations.
Results
A total of 40 studies with 287 transplant patients with metastatic RMS (age range 0 to 32 years) were included in the assessment. We identified 3 non-randomized controlled trials. The 3-year overall survival ranged from 22% to 53% in the transplant groups vs. 18% to 55% in the control groups. Meta-analysis on overall survival in controlled trials showed no difference between treatments. Result of meta-analysis of pooled individual survival data of case series and case reports, and results from uncontrolled studies with aggregate data were in the range of those from controlled data. The risk of bias was high in all studies due to methodological flaws.
Conclusions
HDCT followed by autologous HSCT in patients with RMS remains an experimental treatment. At present, it does not appear justifiable to use this treatment except in appropriately designed controlled trials.
doi:10.1371/journal.pone.0017127
PMCID: PMC3044147  PMID: 21373200
19.  Resveratrol Enhances Antitumor Activity of TRAIL in Prostate Cancer Xenografts through Activation of FOXO Transcription Factor 
PLoS ONE  2010;5(12):e15627.
Background
Resveratrol (3, 4′, 5 tri-hydroxystilbene), a naturally occurring polyphenol, exhibits anti-inflammatory, antioxidant, cardioprotective and antitumor activities. We have recently shown that resveratrol can enhance the apoptosis-inducing potential of TRAIL in prostate cancer cells through multiple mechanisms in vitro. Therefore, the present study was designed to validate whether resveratrol can enhance the apoptosis-inducing potential of TRAIL in a xenograft model of prostate cancer.
Methodology/Principal Findings
Resveratrol and TRAIL alone inhibited growth of PC-3 xenografts in nude mice by inhibiting tumor cell proliferation (PCNA and Ki67 staining) and inducing apoptosis (TUNEL staining). The combination of resveratrol and TRAIL was more effective in inhibiting tumor growth than single agent alone. In xenografted tumors, resveratrol upregulated the expressions of TRAIL-R1/DR4, TRAIL-R2/DR5, Bax and p27/K IP1, and inhibited the expression of Bcl-2 and cyclin D1. Treatment of mice with resveratrol and TRAIL alone inhibited angiogenesis (as demonstrated by reduced number of blood vessels, and VEGF and VEGFR2 positive cells) and markers of metastasis (MMP-2 and MMP-9). The combination of resveratrol with TRAIL further inhibited number of blood vessels in tumors, and circulating endothelial growth factor receptor 2-positive endothelial cells than single agent alone. Furthermore, resveratrol inhibited the cytoplasmic phosphorylation of FKHRL1 resulting in its enhanced activation as demonstrated by increased DNA binding activity.
Conclusions/Significance
These data suggest that resveratrol can enhance the apoptosis-inducing potential of TRAIL by activating FKHRL1 and its target genes. The ability of resveratrol to inhibit tumor growth, metastasis and angiogenesis, and enhance the therapeutic potential of TRAIL suggests that resveratrol alone or in combination with TRAIL can be used for the management of prostate cancer.
doi:10.1371/journal.pone.0015627
PMCID: PMC3011015  PMID: 21209944
20.  Tissue Inhibitor of Metalloproteinase-3 (TIMP-3) Regulates Hematopoiesis and Bone Formation In Vivo 
PLoS ONE  2010;5(9):e13086.
Background
Tissue inhibitor of metalloproteinases-3 (TIMP-3) inhibits matrix metalloproteinases and membrane-bound sheddases. TIMP-3 is associated with the extracellular matrix and is expressed in highly remodeling tissues. TIMP-3 function in the hematopoietic system is unknown.
Methodology/Principal Findings
We now report that TIMP-3 is highly expressed in the endosteal region of the bone marrow (BM), particularly by osteoblasts, endothelial and multipotent mesenchymal stromal cells which are all important cellular components of hematopoietic stem cell (HSC) niches, whereas its expression is very low in mature leukocytes and hematopoietic stem and progenitor cells. A possible role of TIMP-3 as an important niche component was further suggested by its down-regulation during granulocyte colony-stimulating factor-induced mobilization. To further investigate TIMP-3 function, mouse HSC were retrovirally transduced with human TIMP-3 and transplanted into lethally irradiated recipients. TIMP-3 overexpression resulted in decreased frequency of B and T lymphocytes and increased frequency of myeloid cells in blood and BM, increased Lineage-negative Sca-1+KIT+ cell proliferation in vivo and in vitro and increased colony-forming cell trafficking to blood and spleen. Finally, over-expression of human TIMP-3 caused a late onset fatal osteosclerosis.
Conclusions/Significance
Our results suggest that TIMP-3 regulates HSC proliferation, differentiation and trafficking in vivo, as well as bone and bone turn-over, and that TIMP-3 is expressed by stromal cells forming HSC niches within the BM. Thus, TIMP-3 may be an important HSC niche component regulating both hematopoiesis and bone remodeling.
doi:10.1371/journal.pone.0013086
PMCID: PMC2948005  PMID: 20941363
21.  Hematopoietic stem cells: ex-vivo expansion and therapeutic potential for myocardial ischemia 
Despite recent advances in cardiovascular medicine, ischemic heart disease remains the major cause of death in the United States and abroad. Cell-based therapy for degenerative diseases like myocardial ischemia using stem cells is currently under serious investigation. Various types of stem cells are being considered to be candidates for cell transplantation in cell-based therapy. Hematopoietic stem cells are one of the most promising cell types as several studies demonstrated their ability to improve ischemic cardiac functions by enhancing neovascularization and by reducing the total size of scar tissue. However, in order to procure sufficient numbers of functional stem cells, ex-vivo expansion technology became critically important. In this review, we focus on the state-of-the-art ex-vivo technology for the expansion of hematopoietic stem cells, and the underlying mechanisms regulating stem cell self-renewal as well as differentiation.
PMCID: PMC3781726  PMID: 24198511
ischemic heart disease; ex-vivo expansion; hematopoietic stem cells; cytokines; nanofibers
22.  Stem Cell Therapy with Overexpressed VEGF and PDGF Genes Improves Cardiac Function in a Rat Infarct Model 
PLoS ONE  2009;4(10):e7325.
Background
Therapeutic potential was evaluated in a rat model of myocardial infarction using nanofiber-expanded human cord blood derived hematopoietic stem cells (CD133+/CD34+) genetically modified with VEGF plus PDGF genes (VIP).
Methods and Findings
Myocardial function was monitored every two weeks up to six weeks after therapy. Echocardiography revealed time dependent improvement of left ventricular function evaluated by M-mode, fractional shortening, anterior wall tissue velocity, wall motion score index, strain and strain rate in animals treated with VEGF plus PDGF overexpressed stem cells (VIP) compared to nanofiber expanded cells (Exp), freshly isolated cells (FCB) or media control (Media). Improvement observed was as follows: VIP>Exp> FCB>media. Similar trend was noticed in the exercise capacity of rats on a treadmill. These findings correlated with significantly increased neovascularization in ischemic tissue and markedly reduced infarct area in animals in the VIP group. Stem cells in addition to their usual homing sites such as lung, spleen, bone marrow and liver, also migrated to sites of myocardial ischemia. The improvement of cardiac function correlated with expression of heart tissue connexin 43, a gap junctional protein, and heart tissue angiogenesis related protein molecules like VEGF, pNOS3, NOS2 and GSK3. There was no evidence of upregulation in the molecules of oncogenic potential in genetically modified or other stem cell therapy groups.
Conclusion
Regenerative therapy using nanofiber-expanded hematopoietic stem cells with overexpression of VEGF and PDGF has a favorable impact on the improvement of rat myocardial function accompanied by upregulation of tissue connexin 43 and pro-angiogenic molecules after infarction.
doi:10.1371/journal.pone.0007325
PMCID: PMC2752797  PMID: 19809493
23.  Ex Vivo Nanofiber Expansion and Genetic Modification of Human Cord Blood-Derived Progenitor/Stem Cells Enhances Vasculogenesis 
Cell transplantation  2009;18(3):305-318.
The stem cell therapy for treating ischemic diseases is promising; however, the limited availability and compromised quality of progenitor cells in aged and diseased patients limit its therapeutic use. Here we report a nanofiber-based ex vivo stem cell expansion technology and proangiogenic growth factors overexpression of human umbilical cord blood (UCB)-derived progenitor cells to enhance angiogenic potential of therapeutic stem cells. The progenitor cells were expanded ~225-fold on nanofiber-based serum-free ex vivo expansion culture technique without inducing differentiation. The expanded cells express high levels of stem cell homing receptor, CXCR4, and adhesion molecule, LFA-1. The nanofiber-expanded stem cells uptake AcLDL effectively, and migrate efficiently in an in vitro transmigration assay. These expanded cells can also differentiate into endothelial and smooth muscle cells in vitro. In a NOD/SCID mouse hind limb vascular injury model, nanofiber-expanded cells were more effective in blood flow restoration and this effect was further augmented by VEGF164 and PDGF-BB, growth factor overexpression. The data indicate that nanofiber-based ex vivo expansion technology can provide an essential number of therapeutic stem cells. Additionally, proangiogenic growth factors overexpression in progenitor cells can potentially improve autologous or allogeneic stem cell therapy for ischemic diseases.
doi:10.3727/096368909788534870
PMCID: PMC2749501  PMID: 19558779
Human umbilical cord blood; Hematopoietic progenitor stem cells; Proangiogenic growth factors; Nanofibers; Limb ischemia
24.  CD1-mediated γ/δ T Cell Maturation of Dendritic Cells 
The Journal of Experimental Medicine  2002;196(12):1575-1584.
Immature myeloid dendritic cells (DCs) express only low levels of major histocompatibility complex (MHC) class II but express high levels of CD1 a, b, and c antigen-presenting molecules at the cell surface. As Vδ1+ γ/δ T cells are the main tissue subset of γ/δ T cells and they are known to recognize CD1c in the absence of specific foreign antigen recognition, we examined the possible interaction of these T cells with immature DCs. We show that CD1-restricted γ/δ T cells can mediate the maturation of DCs. DC maturation required cell–cell contact and could be blocked by antibodies against CD1c. The maturation process was partially mediated by tumor necrosis factor α. Importantly, immature DCs matured in the presence of lipopolysaccharide and CD1-restricted γ/δ T cells produced bioactive interleukin-12p70. In addition, these DCs were able to efficiently present peptide antigens to naive CD4+ T cells. CD1-restricted γ/δ T cell recognition of immature DCs provides the human immune system with the capacity to rapidly generate a pool of mature DCs early during microbial invasion. This may be an important source of critical host signals for T helper type 1 polarization of antigen-specific naive T cells and the subsequent adaptive immune response.
doi:10.1084/jem.20021515
PMCID: PMC2196072  PMID: 12486100
T cell antigen receptors γ/δ; CD1; dendritic cells; antigen-presenting cell; tumor necrosis factor
25.  Antibacterial effect of human Vγ2Vδ2 T cells in vivo 
Journal of Clinical Investigation  2001;108(9):1349-1357.
Vγ2Vδ2 cells, a class of T cells found only in primates, are reactive to nonpeptide organophosphate and alkylamine antigens secreted by bacteria and parasites. These cells make up 2-5% percent of human peripheral blood T cells but expand to make up 8–60% of peripheral blood T cells during bacterial and parasitic infections. We show here, using a chimeric severe combined immunodeficiency (SCID) mouse (hu-SCID) model, that human Vγ2Vδ2 T cells mediate resistance to extracellular gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli and Morganella morganii) bacteria, as assessed by survival, body weight, bacterial loads, and histopathology. Surprisingly, this bacterial resistance was evident 1 day after infection, and bacteria were cleared well before γδ T cell expansion was detected 6 days after infection. Decreased resistance in Vδ2 T cell–depleted hu-SCID mice correlated with decreased serum IFN-γ titers. Intravenous treatment of infected, reconstituted hu-SCID mice with pamidronate, a human Vγ2Vδ2 T cell–specific aminobisphosphonate antigen, markedly increased the in vivo antibacterial effect of Vγ2Vδ2 T cells. Therefore, this large pool of antigen-specific, yet immediately reactive memory human Vγ2Vδ2 T cells is likely to be an important mediator of resistance against extracellular bacterial infection and may bridge the gap between innate and acquired immunity.
PMCID: PMC209444  PMID: 11696580

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