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1.  Spheroid Culture for Enhanced Differentiation of Human Embryonic Stem Cells to Hepatocyte-Like Cells 
Stem Cells and Development  2013;23(2):124-131.
Stem cell-derived hepatocyte-like cells hold great potential for the treatment of liver disease and for drug toxicity screening. The success of these applications hinges on the generation of differentiated cells with high liver specific activities. Many protocols have been developed to guide human embryonic stem cells (hESCs) to differentiate to the hepatic lineage. Here we report cultivation of hESCs as three-dimensional aggregates that enhances their differentiation to hepatocyte-like cells. Differentiation was first carried out in monolayer culture for 20 days. Subsequently cells were allowed to self-aggregate into spheroids. Significantly higher expression of liver-specific transcripts and proteins, including Albumin, phosphoenolpyruvate carboxykinase, and asialoglycoprotein receptor 1 was observed. The differentiated phenotype was sustained for more than 2 weeks in the three-dimensional spheroid culture system, significantly longer than in monolayer culture. Cells in spheroids exhibit morphological and ultrastructural characteristics of primary hepatocytes by scanning and transmission electron microscopy in addition to mature functions, such as biliary excretion of metabolic products and cytochrome P450 activities. This three-dimensional spheroid culture system may be appropriate for generating high quality, functional hepatocyte-like cells from ESCs.
doi:10.1089/scd.2013.0097
PMCID: PMC3887452  PMID: 24020366
2.  Autologous circulating angiogenic cells treated with osteopontin and delivered via a collagen scaffold enhance wound healing in the alloxan-induced diabetic rabbit ear ulcer model 
Introduction
Diabetic foot ulceration is the leading cause of amputation in people with diabetes mellitus. Peripheral vascular disease is present in the majority of patients with diabetic foot ulcers. Despite standard treatments there exists a high amputation rate. Circulating angiogenic cells previously known as early endothelial progenitor cells are derived from peripheral blood and support angiogenesis and vasculogenesis, providing a potential topical treatment for non-healing diabetic foot ulcers.
Methods
A scaffold fabricated from Type 1 collagen facilitates topical cell delivery to a diabetic wound. Osteopontin is a matricellular protein involved in wound healing and increases the angiogenic potential of circulating angiogenic cells. A collagen scaffold seeded with circulating angiogenic cells was developed. Subsequently the effect of autologous circulating angiogenic cells that were seeded in a collagen scaffold and topically delivered to a hyperglycemic cutaneous wound was assessed. The alloxan-induced diabetic rabbit ear ulcer model was used to determine healing in response to the following treatments: collagen seeded with autologous circulating angiogenic cells exposed to osteopontin, collagen seeded with autologous circulating angiogenic cells, collagen alone and untreated wound. Stereology was used to assess angiogenesis in wounds.
Results
The cells exposed to osteopontin and seeded on collagen increased percentage wound closure as compared to other groups. Increased angiogenesis was observed with the treatment of collagen and collagen seeded with circulating angiogenic cells.
Conclusions
These results demonstrate that topical treatment of full thickness cutaneous ulcers with autologous circulating angiogenic cells increases wound healing. Cells exposed to the matricellular protein osteopontin result in superior wound healing. The wound healing benefit is associated with a more efficient vascular network. This topical therapy provides a potential novel therapy for the treatment of non-healing diabetic foot ulcers in humans.
doi:10.1186/scrt388
PMCID: PMC4054999  PMID: 24444259
3.  Proteomic Analysis of Highly Prevalent Amyloid A Amyloidosis Endemic to Endangered Island Foxes 
PLoS ONE  2014;9(11):e113765.
Amyloid A (AA) amyloidosis is a debilitating, often fatal, systemic amyloid disease associated with chronic inflammation and persistently elevated serum amyloid A (SAA). Elevated SAA is necessary but not sufficient to cause disease and the risk factors for AA amyloidosis remain poorly understood. Here we identify an extraordinarily high prevalence of AA amyloidosis (34%) in a genetically isolated population of island foxes (Urocyon littoralis) with concurrent chronic inflammatory diseases. Amyloid deposits were most common in kidney (76%), spleen (58%), oral cavity (45%), and vasculature (44%) and were composed of unbranching, 10 nm in diameter fibrils. Peptide sequencing by mass spectrometry revealed that SAA peptides were dominant in amyloid-laden kidney, together with high levels of apolipoprotein E, apolipoprotein A-IV, fibrinogen-α chain, and complement C3 and C4 (false discovery rate ≤0.05). Reassembled peptide sequences showed island fox SAA as an 111 amino acid protein, most similar to dog and artic fox, with 5 unique amino acid variants among carnivores. SAA peptides extended to the last two C-terminal amino acids in 5 of 9 samples, indicating that near full length SAA was often present in amyloid aggregates. These studies define a remarkably prevalent AA amyloidosis in island foxes with widespread systemic amyloid deposition, a unique SAA sequence, and the co-occurrence of AA with apolipoproteins.
doi:10.1371/journal.pone.0113765
PMCID: PMC4245998  PMID: 25429466
4.  Robust, Efficient, and Practical Electrogene Transfer Method for Human Mesenchymal Stem Cells Using Square Electric Pulses 
Human Gene Therapy Methods  2013;24(5):289-297.
Abstract
Mesenchymal stem cells (MSCs) are multipotent nonhematopoietic cells with the ability to differentiate into various specific cell types, thus holding great promise for regenerative medicine. Early clinical trials have proven that MSC-based therapy is safe, with possible efficacy in various diseased states. Moreover, genetic modification of MSCs to improve their function can be safely achieved using electrogene transfer. We previously achieved transfection efficiencies of up to 32% with preserved viability in rat MSCs. In this study, we further improved the transfection efficiency and transgene expression in human MSCs (hMSCs), while preserving the cells viability and ability to differentiate into osteoblasts and adipocytes by increasing the plasmid concentration and altering the osmotic pressure of the electrotransfer buffer. Using a square-wave electric pulse generator, we achieved a transfection efficiency of more than 80%, with around 70% viability and a detectable transgene expression of up to 30 days. Moreover, we demonstrated that this transfection efficiency can be reproduced reliably on two different sources of hMSCs: the bone marrow and adipose tissue. We also showed that there was no significant donor variability in terms of their transfection efficiency and viability. The cell confluency before electrotransfer had no significant effect on the transfection efficiency and viability. Cryopreservation of transfected cells maintained their transgene expression and viability upon thawing. In summary, we are reporting a robust, safe, and efficient protocol of electrotransfer for hMSCs with several practical suggestions for an optimal use of genetically engineered hMSCs for clinical application.
Liew and colleagues employ square-wave electric pulses to achieve efficient gene transfer in human mesenchymal stem cells (hMSCs). Robust gene transfer is reproducible across donors and with both bone marrow- and adipose-derived hMSCs. Transfected cells exhibit high viability, normal differentiation capacity, and durable transgene expression.
doi:10.1089/hgtb.2012.159
PMCID: PMC3798228  PMID: 23931158
5.  Mesenchymal Stem Cell Survival in the Infarcted Heart Is Enhanced by Lentivirus Vector-Mediated Heat Shock Protein 27 Expression 
Human Gene Therapy  2013;24(10):840-851.
Abstract
Mesenchymal stem cell (MSC) therapy offers the potential to promote recovery after myocardial infarction (MI). However, therapeutic efficacy may be limited by poor survival and retention of transplanted cells. A combination of gene and cell therapy has the capacity to prevent donor cell death and augment the reparative and regenerative effects of cell transfer. The present study investigates the effect of exogenous heat shock protein 27 (Hsp27) expression in MSCs in an in vitro model of ischemia and in an in vivo rat MI model and aims to determine if this could enhance the therapeutic benefit associated with cell delivery. Hsp27 overexpression by lentivirus vector modification resulted in increased MSC survival in vitro and in vivo. Furthermore, decreased apoptosis in the infarcted tissue and improved cardiac function was observed in the Hsp27 group, enhancing the therapeutic effect of MSCs. Together, these data demonstrate that ex vivo genetic modification—specifically Hsp27 overexpression—offers the possibility of enhancing the efficacy of MSC therapy in MI.
McGinley and colleagues demonstrate that mesenchymal stem cells (MSCs) can be rendered resistant to ischemia and hypoxia in vitro through lentivirus-mediated overexpression of heat shock protein 27 (Hsp27). In a rat model of myocardial ischemia, Hsp27-modified MSCs exhibit improved engraftment compared with unmodified cells with associated benefits in cardiac function.
doi:10.1089/hum.2011.009
PMCID: PMC3787467  PMID: 23987185
6.  Abnormal Infant Islet Morphology Precedes Insulin Resistance in PCOS-Like Monkeys 
PLoS ONE  2014;9(9):e106527.
Polycystic ovary syndrome (PCOS) is prevalent in reproductive-aged women and confounded by metabolic morbidities, including insulin resistance and type 2 diabetes. Although the etiology of PCOS is undefined, contribution of prenatal androgen (PA) exposure has been proposed in a rhesus monkey model as premenopausal PA female adults have PCOS-like phenotypes in addition to insulin resistance and decreased glucose tolerance. PA female infants exhibit relative hyperinsulinemia, suggesting prenatal sequelae of androgen excess on glucose metabolism and an antecedent to future metabolic disease. We assessed consequences of PA exposure on pancreatic islet morphology to identify evidence of programming on islet development. Islet counts and size were quantified and correlated with data from intravenous glucose tolerance tests (ivGTT) obtained from dams and their offspring. Average islet size was decreased in PA female infants along with corresponding increases in islet number, while islet fractional area was preserved. Infants also demonstrated an increase in both the proliferation marker Ki67 within islets and the beta to alpha cell ratio suggestive of enhanced beta cell expansion. PA adult females have reduced proportion of small islets without changes in proliferative or apoptotic markers, or in beta to alpha cell ratios. Together, these data suggest in utero androgen excess combined with mild maternal glucose intolerance alter infant and adult islet morphology, implicating deviant islet development. Marked infant, but subtle adult, morphological differences provide evidence of islet post-natal plasticity in adapting to changing physiologic demands: from insulin sensitivity and relative hypersecretion to insulin resistance and diminished insulin response to glucose in the mature PCOS-like phenotype.
doi:10.1371/journal.pone.0106527
PMCID: PMC4160158  PMID: 25207967
7.  Predator and prey activity levels jointly influence the outcome of long-term foraging bouts 
Behavioral Ecology  2013;24(5):1205-1210.
Lay summary
Animals exhibit “behavioral types” (akin to human personalities) where individuals differ consistently on traits like activity which may influence the predators it encounters and the prey it captures. Here we demonstrate that active jumping spiders are more likely to encounter and consume inactive crickets and vice versa. This presents a potential explanation for the persistence of behavioral types in natural populations, as behavioral variation in one trophic level maintains variation in the associated level.
Consistent interindividual differences in behavior (i.e., “behavioral types”) may be a key factor in determining the outcome of species interactions. Studies that simultaneously account for the behavioral types of individuals in multiple interacting species, such as predator–prey systems, may be particularly strong predictors of ecological outcomes. Here, we test the predator–prey locomotor crossover hypothesis, which predicts that active predators are more likely to encounter and consume prey with the opposing locomotor tendency. We test this hypothesis using intraspecific behavioral variation in both a predator and prey species as predictors of foraging outcomes. We use the old field jumping spider, Phidippus clarus (Araneae, Salticidae), and the house cricket, Acheta domesticus (Orthoptera, Gryllidae), as a model predator–prey system in laboratory mesocosm trials. Stable individual differences in locomotor tendencies were identified in both P. clarus and A. domesticus, and the outcome of foraging bouts depended neither on the average activity level of the predator nor on the average activity level of prey. Instead, an interaction between the activity level of spiders and crickets predicted spider foraging success and prey survivorship. Consistent with the locomotor crossover hypothesis, predators exhibiting higher activity levels consumed more prey when in an environment containing low-activity prey items and vice versa. This study highlights 1) the importance of intraspecific variation in determining the outcome of predator–prey interactions and 2) that acknowledging behavioral variation in only a single species may be insufficient to characterize the performance consequences of intraspecific trait variants.
doi:10.1093/beheco/art052
PMCID: PMC3739417  PMID: 23935257
behavioral syndrome; foraging mode; intraspecific variation; personality; predator–prey interaction.
8.  Sensory Neuron Fates Are Distinguished by a Transcriptional Switch that Regulates Dendrite Branch Stabilization 
Neuron  2013;79(2):266-280.
SUMMARY
Sensory neurons adopt distinct morphologies and functional modalities to mediate responses to specific stimuli. Transcription factors and their downstream effectors orchestrate this outcome but are incompletely defined. Here, we show that different classes of mechanosensory neurons in C. elegans are distinguished by the combined action of the transcription factors MEC-3, AHR-1, and ZAG-1. Low levels of MEC-3 specify the elaborate branching pattern of PVD nociceptors, whereas high MEC-3 is correlated with the simple morphology of AVM and PVM touch neurons. AHR-1 specifies AVM touch neuron fate by elevating MEC-3 while simultaneously blocking expression of nociceptive genes such as the MEC-3 target, the claudin-like membrane protein HPO-30, that promotes the complex dendritic branching pattern of PVD. ZAG-1 exercises a parallel role to prevent PVM from adopting the PVD fate. The conserved dendritic branching function of the Drosophila AHR-1 homolog, Spineless, argues for similar pathways in mammals.
doi:10.1016/j.neuron.2013.05.009
PMCID: PMC3795438  PMID: 23889932
9.  Topical Administration of Allogeneic Mesenchymal Stromal Cells Seeded in a Collagen Scaffold Augments Wound Healing and Increases Angiogenesis in the Diabetic Rabbit Ulcer 
Diabetes  2013;62(7):2588-2594.
There is a critical clinical need to develop therapies for nonhealing diabetic foot ulcers. Topically applied mesenchymal stromal cells (MSCs) provide a novel treatment to augment diabetic wound healing. A central pathological factor in nonhealing diabetic ulcers is an impaired blood supply. It was hypothesized that topically applied allogeneic MSCs would improve wound healing by augmenting angiogenesis. Allogeneic nondiabetic bone-marrow derived MSCs were seeded in a collagen scaffold. The cells were applied to a full-thickness cutaneous wound in the alloxan-induced diabetic rabbit ear ulcer model in a dose escalation fashion. Percentage wound closure and angiogenesis at 1 week was assessed using wound tracings and stereology, respectively. The topical application of 1,000,000 MSCs on a collagen scaffold demonstrated increased percentage wound closure when compared with lower doses. The collagen and collagen seeded with MSCs treatments result in increased angiogenesis when compared with untreated wounds. An improvement in wound healing as assessed by percentage wound closure was observed only at the highest cell dose. This cell-based therapy provides a novel therapeutic strategy for increasing wound closure and augmenting angiogenesis, which is a central pathophysiological deficit in the nonhealing diabetic foot ulcer.
doi:10.2337/db12-1822
PMCID: PMC3712062  PMID: 23423568
10.  Gene Therapy for Type 1 Diabetes Moves a Step Closer to Reality 
Diabetes  2013;62(5):1396-1397.
doi:10.2337/db13-0348
PMCID: PMC3636656  PMID: 23613564
11.  Mesenchymal Stem Cell (MSC) mediated delivery of the Sodium Iodide Symporter (NIS) supports radionuclide imaging and treatment of breast cancer 
Stem cells (Dayton, Ohio)  2011;29(7):1149-1157.
Mesenchymal Stem Cells (MSCs) migrate specifically to tumors in vivo, and coupled with their capacity to bypass immune surveillance, are attractive vehicles for tumor-targeted delivery of therapeutic agents. This study aimed to introduce MSC-mediated expression of the sodium iodide symporter (NIS) for imaging and therapy of breast cancer. Tumor bearing animals received an intravenous or intratumoral injection of NIS expressing MSCs (MSC-NIS), followed by 99mTcO4- imaging 3-14Days (D) later using a BazookaSPECT γ-camera. Tissue was harvested for analysis of hNIS expression by RQPCR. Therapy animals received an intraperitoneal injection of 131I or saline 14D following injection of MSC-NIS, and tumor volume was monitored for 8 weeks. BazookaSPECT imaging following injection of MSC-NIS revealed an image of animal intestines and chest area at D3, with a weak tumor image also visible. By D14, the tumor was visible with a significant reduction in radionuclide accumulation in non-target tissue observed. hNIS gene expression was detected in the intestines, heart, lungs and tumor at early timepoints but later depleted in non-target tissues and persisted at the tumor site. Based on imaging/biodistribution data, animals received a therapeutic dose of 131I 14D following MSC-NIS injection. This resulted in a significant reduction in tumor growth (Mean ± SEM, 236 ± 62mm3 versus 665 ± 204 mm3 in controls). The ability to noninvasively track MSC migration and transgene expression in real time prior to therapy is a major advantage to this strategy. This promising data supports the feasibility of this approach as a novel therapy for breast cancer.
doi:10.1002/stem.665
PMCID: PMC3998644  PMID: 21608083
Sodium Iodide Symporter (NIS); Mesenchymal Stem Cell (MSC); Breast Cancer; Gene therapy; In vivo Imaging; Radiotherapy
12.  Mesenchymal Stem Cell-Based Treatment for Microvascular and Secondary Complications of Diabetes Mellitus 
The worldwide increase in the prevalence of Diabetes mellitus (DM) has highlighted the need for increased research efforts into treatment options for both the disease itself and its associated complications. In recent years, mesenchymal stromal cells (MSCs) have been highlighted as a new emerging regenerative therapy due to their multipotency but also due to their paracrine secretion of angiogenic factors, cytokines, and immunomodulatory substances. This review focuses on the potential use of MSCs as a regenerative medicine in microvascular and secondary complications of DM and will discuss the challenges and future prospects of MSCs as a regenerative therapy in this field. MSCs are believed to have an important role in tissue repair. Evidence in recent years has demonstrated that MSCs have potent immunomodulatory functions resulting in active suppression of various components of the host immune response. MSCs may also have glucose lowering properties providing another attractive and unique feature of this therapeutic approach. Through a combination of the above characteristics, MSCs have been shown to exert beneficial effects in pre-clinical models of diabetic complications prompting initial clinical studies in diabetic wound healing and nephropathy. Challenges that remain in the clinical translation of MSC therapy include issues of MSC heterogeneity, optimal mode of cell delivery, homing of these cells to tissues of interest with high efficiency, clinically meaningful engraftment, and challenges with cell manufacture. An issue of added importance is whether an autologous or allogeneic approach will be used. In summary, MSC administration has significant potential in the treatment of diabetic microvascular and secondary complications but challenges remain in terms of engraftment, persistence, tissue targeting, and cell manufacture
doi:10.3389/fendo.2014.00086
PMCID: PMC4047679  PMID: 24936198
mesenchymal stromal cell; MSC; diabetes; microvascular complication; retinopathy; nephropathy; neuropathy
13.  Phase II Study of Dasatinib (BMS-354825) in Patients With Metastatic Adenocarcinoma of the Pancreas 
The Oncologist  2013;18(10):1091-1092.
Background.
Src, EphA2, and platelet-derived growth factor receptors α and β are dysregulated in pancreatic ductal adenocarcinoma (PDAC). Dasatinib is an oral multitarget tyrosine kinase inhibitor that targets BCR-ABL, c-Src, c-KIT, platelet-derived growth factor receptor β, and EphA2. We conducted a phase II, single-arm study of dasatinib as first-line therapy in patients with metastatic PDAC.
Methods.
Dasatinib (100 mg twice a day, later reduced to 70 mg twice a day because of toxicities) was orally administered continuously on a 28-day cycle. The primary endpoint was overall survival (OS). Response was measured using the Response Evaluation Criteria in Solid Tumors. Circulating tumor cells (CTCs) were also collected.
Results.
Fifty-one patients enrolled in this study. The median OS was 4.7 months (95% confidence interval [CI]: 2.8–6.9 months). Median progression-free survival was 2.1 months (95% CI: 1.6–3.2 months). In 34 evaluable patients, the best response achieved was stable disease in 10 patients (29.4%). One patient had stable disease while on treatment for 20 months. The most common nonhematologic toxicities were fatigue and nausea. Edema and pleural effusions occurred in 29% and 6% of patients, respectively. The number of CTCs did not correlate with survival.
Conclusion.
Single-agent dasatinib does not have clinical activity in metastatic PDAC.
doi:10.1634/theoncologist.2013-0255
PMCID: PMC3805150  PMID: 24072218
14.  Therapeutic potential for mesenchymal stem cell transplantation in critical limb ischemia 
The therapeutic potential of mesenchymal stem cell (MSC) transplantation for the treatment of ischemic conditions such as coronary artery disease, peripheral arterial disease, and stroke has been explored in animal models and early-phase clinical trials. A substantial database documents the safety profile of MSC administration to humans in a large number of disease states. The mechanism of the therapeutic effect of MSC transplantation in ischemic disease has been postulated to be due to paracrine, immunomodulatory, and differentiation effects. This review provides an overview of the potential role of MSC-based therapy for critical limb ischemia (CLI), the comparison of MSC cellular therapy with angiogenesis gene therapy in CLI, and the proposed mechanism of action of MSC therapy. Preclinical efficacy data in animal models of hindlimb ischemia, current early-phase human trial data, and considerations for future MSC-based therapy in CLI will also be discussed.
doi:10.1186/scrt119
PMCID: PMC3580466  PMID: 22846185
15.  CD40 ligand is necessary and sufficient to support primary diffuse large B-cell lymphoma cells in culture: a tool for in vitro preclinical studies with primary B-cell malignancies 
Leukemia & lymphoma  2012;53(7):1390-1398.
Established cell lines are utilized extensively to study tumor biology and preclinical therapeutic development; however, they may not accurately recapitulate the heterogeneity of their corresponding primary disease. B-cell tumor cells are especially difficult to maintain under conventional culture conditions, limiting access to samples that faithfully represent this disease for preclinical studies. Here, we used primary canine diffuse large B-cell lymphoma to establish a culture system that reliably supports the growth of these cells. CD40 ligand, either expressed by feeder cells or provided as a soluble two-trimeric form, was sufficient to support primary lymphoma cells in vitro. The tumor cells retained their original phenotype, clonality and known karyotypic abnormalities after extended expansion in culture. Finally, we illustrate the utility of the feeder cell-free culture system for comparable assessment of cytotoxicity using dog and human B-cell malignancies. We conclude this system has broad applications for in vitro preclinical development for B-cell malignancies.
doi:10.3109/10428194.2011.654337
PMCID: PMC3727651  PMID: 22229753
Diffuse large B-cell lymphoma; CD40L; Primary cell culture; Canine model; Cytotoxicity assay
16.  Three years of Stem Cell Research & Therapy 
doi:10.1186/scrt196
PMCID: PMC3706755  PMID: 23635482
17.  Stem Cell Research & Therapy in 2012 
doi:10.1186/scrt107
PMCID: PMC3392776  PMID: 22548746
18.  Inhibition of pulmonary nuclear factor kappa-B decreases the severity of acute Escherichia coli pneumonia but worsens prolonged pneumonia 
Critical Care  2013;17(2):R82.
Introduction
Nuclear factor (NF)-κB is central to the pathogenesis of inflammation in acute lung injury, but also to inflammation resolution and repair. We wished to determine whether overexpression of the NF-κB inhibitor IκBα could modulate the severity of acute and prolonged pneumonia-induced lung injury in a series of prospective randomized animal studies.
Methods
Adult male Sprague-Dawley rats were randomized to undergo intratracheal instillation of (a) 5 × 109 adenoassociated virus (AAV) vectors encoding the IκBα transgene (5 × 109 AAV-IκBα); (b) 1 × 1010 AAV-IκBα; (c) 5 × 1010 AAV-IκBα; or (d) vehicle alone. After intratracheal inoculation with Escherichia coli, the severity of the lung injury was measured in one series over a 4-hour period (acute pneumonia), and in a second series after 72 hours (prolonged pneumonia). Additional experiments examined the effects of IκBα and null-gene overexpression on E. coli-induced and sham pneumonia.
Results
In acute pneumonia, IκBα dose-dependently decreased lung injury, improving arterial oxygenation and lung static compliance, reducing alveolar protein leak and histologic injury, and decreasing alveolar IL-1β concentrations. Benefit was maximal at the intermediate (1 × 1010) IκBα vector dose; however, efficacy was diminished at the higher (5 × 1010) IκBα vector dose. In contrast, IκBα worsened prolonged pneumonia-induced lung injury, increased lung bacterial load, decreased lung compliance, and delayed resolution of the acute inflammatory response.
Conclusions
Inhibition of pulmonary NF-κB activity reduces early pneumonia-induced injury, but worsens injury and bacterial load during prolonged pneumonia.
doi:10.1186/cc12696
PMCID: PMC4056114  PMID: 23622108
Acute lung injury; inhibitory kappa-B alpha; rat; acute respiratory distress syndrome; bacteria; pneumonia; gene therapy
20.  Brief Report: Phase II Trial of Rebeccamycin Analogue, a Dual Topoisomerase I and II Inhibitor, in Relapsed “Sensitive” Small Cell Lung Cancer 
Journal of Thoracic Oncology  2012;7(4):751-754.
Relapsed small cell lung cancer (SCLC) carries a poor prognosis. Toposiomerase I and II inhibitors and DNA damaging agents are considered amongst the most active agents against SCLC. Rebeccamaycin analogue (RA, Becatacarin) is an anti-tumor antibiotic with inhibitory activity against both topoisomerase I and II as well as DNA intercalating properties. We performed a phase II trial of RA in relapsed, sensitive SCLC with the primary endpoint of response rate. Patients with previously treated SCLC who relapsed more than 60 days after completion of first-line chemotherapy were treated with RA administered i.v. at a dose of 140 mg/m2 on days 1–5 of 21 day cycles for a maximum of 6 cycles. Eligibility included ECOG PS 0–2 and adequate organ function. A 2-stage design was employed. Twenty evaluable patients were enrolled. Median age was 61 years. Two (10%) patients had a partial response and six had stable disease. The clinical benefit rate (CBR) was 40% (95% CI 23–64%). The median progression free survival was 2 months (95% CI 1.2–5.2 mo). The median survival was 6.7 months (95% CI: 3.3–8.0 months). No treatment-related deaths occurred. Grade 4 neutropenia and thrombocytopenia occurred in 23% and 14% of patients respectively. In conclusion, RA has single-agent activity in relapsed, sensitive SCLC with manageable toxicities but is unlikely to provide any superiority compared to existing agents for this disease.
doi:10.1097/JTO.0b013e31824abca2
PMCID: PMC3310884  PMID: 22425925
21.  A poisonous surprise under the coat of the African crested rat 
Plant toxins are sequestered by many animals and the toxicity is frequently advertised by aposematic displays to deter potential predators. Such ‘unpalatability by appropriation’ is common in many invertebrate groups and also found in a few vertebrate groups. However, potentially lethal toxicity by acquisition has so far never been reported for a placental mammal. Here, we describe complex morphological structures and behaviours whereby the African crested rat, Lophiomys imhausi, acquires, dispenses and advertises deterrent toxin. Roots and bark of Acokanthera schimperi (Apocynaceae) trees are gnawed, masticated and slavered onto highly specialized hairs that wick up the compound, to be delivered whenever the animal is bitten or mouthed by a predator. The poison is a cardenolide, closely resembling ouabain, one of the active components in a traditional African arrow poison long celebrated for its power to kill elephants.
doi:10.1098/rspb.2011.1169
PMCID: PMC3248729  PMID: 21813554
Lophiomys; Acokanthera; ouabain; toxicity; aposematic; mammal
22.  Arginase Treatment Prevents the Recovery of Canine Lymphoma and Osteosarcoma Cells Resistant to the Toxic Effects of Prolonged Arginine Deprivation 
PLoS ONE  2013;8(1):e54464.
Rapidly growing tumor cells require a nutrient-rich environment in order to thrive, therefore, restricting access to certain key amino acids, such as arginine, often results in the death of malignant cells, which frequently display defective cell cycle check-point control. Healthy cells, by contrast, become quiescent and remain viable under arginine restriction, displaying full recovery upon return to arginine-rich conditions. The use of arginase therapy to restrict available arginine for selectively targeting malignant cells is currently under investigation in human clinical trials. However, the suitability of this approach for veterinary uses is unexplored. As a prelude to in vivo studies in canine malignancies, we examined the in vitro effects of arginine-deprivation on canine lymphoid and osteosarcoma cell lines. Two lymphoid and 2 osteosarcoma cell lines were unable to recover following 6 days of arginine deprivation, but all remaining cell lines displayed full recovery upon return to arginine-rich culture conditions. These remaining cell lines all proved susceptible to cell death following the addition of arginase to the cultures. The lymphoid lines were particularly sensitive to arginase, becoming unrecoverable after just 3 days of treatment. Two of the osteosarcoma lines were also susceptible over this time-frame; however the other 3 lines required 6–8 days of arginase treatment to prevent recovery. In contrast, adult progenitor cells from the bone marrow of a healthy dog were able to recover fully following 9 days of culture in arginase. Over 3 days in culture, arginase was more effective than asparaginase in inducing the death of lymphoid lines. These results strongly suggest that short-term arginase treatment warrants further investigation as a therapy for lymphoid malignancies and osteosarcomas in dogs.
doi:10.1371/journal.pone.0054464
PMCID: PMC3554772  PMID: 23365669
24.  A comparison of the efficacy of transplantation of bone marrow-derived mesenchymal stem cells and unrestricted somatic stem cells on outcome after acute myocardial infarction 
Introduction
A number of questions remain unanswered in the field of cell therapy for acute myocardial infarction, including what is the optimal cell type, and can therapeutic efficacy be enhanced by conditioning regimens. In this study, we sought to address these questions by directly comparing the effect of bone marrow-derived mesenchymal stem cells and unrestricted somatic stem cells delivered 24 hours post-myocardial infarction and by determining if the therapeutic efficacy of unrestricted somatic stem cells could be enhanced by exposing the cells to guiding factors before cell transplantation.
Methods
Unrestricted somatic stem cells were guided by exposure to 50 ng/mL basic fibroblast growth factor, 20 ng/mL hepatocyte growth factor and 20 ng/mL bone morphogenetic protein-2 for 24 hours. Using a Sprague-Dawley rat model of acute myocardial infarction, we transplanted cells by intramyocardial injection 24 hours post-myocardial infarction. Cardiac function was serially measured using echocardiography, and histological analyses of infarct morphology, angiogenesis and apoptosis were obtained. Transcriptomic and proteomic changes were assessed using microarray and real-time quantitative PCR.
Results
When assessed 28 days after the myocardial infarction, the delivery of mesenchymal stem cells 24 hours post-myocardial infarction did not improve ejection fraction (P = 0.19), and did not prevent the decline in ejection fraction observed in the absence of cell therapy (P = 0.17). The administration of unrestricted somatic stem cells also did not improve ejection fraction (P = 0.11), but did prevent a further decline in ejection fraction (P = 0.001). Delivery of guided unrestricted somatic stem cells significantly improved ejection fraction (P = 0.03). Guided unrestricted somatic stem cells restored function to a greater extent than mesenchymal stem cells (P = 0.03). The infarct area (P = 0.2), apoptosis (P = 0.07) and angiogenesis (P = 0.09) did not differ between groups. Microarray analysis revealed that, following pre-implantation guiding, the gene groupings of mitosis, signalling and angiogenesis were highly overrepresented, mediators of apoptosis were overrepresented, and cardiomyocyte-associated genes were not differentially expressed.
Conclusions
These results suggest that guided unrestricted somatic stem cells have a moderate capacity to repair cardiac damage and that they are more effective than mesenchymal stem cells in restoring cardiac function after a myocardial infarction. The mechanism of the benefit was not fully elucidated in this study, but these observations may be mediated by favorable dysregulation of angiogenic and apoptotic gene groupings.
doi:10.1186/scrt127
PMCID: PMC3580427  PMID: 22974654
Cardiac failure; cardiac repair; guiding; mesenchymal stem cell; myocardial infarction; pre-conditioned; stem cell; umbilical cord; unrestricted somatic stem cell
25.  Enhanced Differentiation of Adult Bone Marrow-Derived Stem Cells to Liver Lineage in Aggregate Culture 
Tissue Engineering. Part A  2011;17(17-18):2331-2341.
Hepatocyte-like cells derived from stem cells hold great potential for clinical and pharmaceutical applications, including high-throughput drug toxicity screening. We report a three-dimensional aggregate culture system for the directed differentiation of adult rat bone marrow-derived stem cells, rat multipotent adult progenitor cells, to hepatocyte-like cells. Compared to adherent monolayer cultures, differentiation in the aggregate culture system resulted in significantly higher expression level of liver-specific transcripts, including an increased albumin mRNA level, and higher levels of albumin and urea secretion. This coincides with the presence of significantly more cells that express intracellular albumin at levels found in primary hepatocytes. The differentiated cell aggregates exhibited cytochrome P450-mediated ethoxyresorufin-O-dealkylation and pentoxyresorufin-O-dealkylation activity. Consistent with these increased mature functions, cells within the aggregates were shown to have many ultrastructural features of mature hepatocytes by transmission electron microscopy. With the scalability of the aggregate culture system and the enhanced differentiation capability, this system may facilitate translation of generating hepatocytes from stem cells to technology.
doi:10.1089/ten.tea.2010.0667
PMCID: PMC3161102  PMID: 21548835

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