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1.  In Vitro Platforms for Evaluating Liver Toxicity 
The liver is a heterogeneous organ with many vital functions, including metabolism of pharmaceutical drugs and is highly susceptible to injury from these substances. The etiology of drug induced liver disease is still debated although generally regarded as a continuum between an activated immune response and hepatocyte metabolic dysfunction, most often resulting from an intermediate reactive metabolite. This debate stems from the fact that current animal and in vitro models provide limited physiologically relevant information and their shortcomings have resulted in ‘silent’ hepatotoxic drugs being introduced into clinical trials, garnering huge financial losses for drug companies through withdrawals and late stage clinical failures. As we advance our understanding into the molecular processes leading to liver injury, it is increasingly clear that a) the pathologic lesion is not only due to liver parenchyma but is also due to the interactions between the hepatocytes and the resident liver immune cells, stellate cells and endothelial cells; and, b) animal models do not reflect the human cell interactions. Therefore, a predictive human, in vitro model must address the interactions between the major human liver cell types and measure key determinants of injury such as the dosage and metabolism of the drug, the stress response, cholestatic effect, and the immune and fibrotic response. In this mini-review, we first discuss the current state of macro-scale in vitro liver culture systems with examples that have been commercialized. We then introduce the paradigm of microfluidic culture systems that aim to mimic the liver with physiologically relevant dimensions, cellular structure, perfusion and mass transport by taking advantage of micro and nanofabrication technologies. We review the most prominent liver-on-a-chip platforms in terms of their physiological relevance and drug response. We conclude with a commentary on other critical advances such as the deployment of fluorescence-based biosensors to identify relevant toxicity pathways, as well as computational models to create a predictive tool.
doi:10.1177/1535370214531872
PMCID: PMC4156546  PMID: 24764241
Drug Induced Liver Injury; Liver on chip; Hepatotoxicity; High Content Screening; Predictive Modeling
2.  Successful Supercooled Liver Storage for 4 Days 
Nature medicine  2014;20(7):790-793.
The realization of long–term human organ preservation will have groundbreaking effects on the current practice of transplantation. Herein we present a novel technique based on sub–zero non–freezing tissue preservation and extracorporeal machine perfusion that allows transplantation of rat livers preserved for up to 4 days, thereby tripling the viable preservation duration.
doi:10.1038/nm.3588
PMCID: PMC4141719  PMID: 24973919
3.  Towards a three-dimensional microfluidic liver platform for predicting drug efficacy and toxicity in humans 
Stem Cell Research & Therapy  2013;4(Suppl 1):S16.
Although the process of drug development requires efficacy and toxicity testing in animals prior to human testing, animal models have limited ability to accurately predict human responses to xenobiotics and other insults. Societal pressures are also focusing on reduction of and, ultimately, replacement of animal testing. However, a variety of in vitro models, explored over the last decade, have not been powerful enough to replace animal models. New initiatives sponsored by several US federal agencies seek to address this problem by funding the development of physiologically relevant human organ models on microscopic chips. The eventual goal is to simulate a human-on-a-chip, by interconnecting the organ models, thereby replacing animal testing in drug discovery and development. As part of this initiative, we aim to build a three-dimensional human liver chip that mimics the acinus, the smallest functional unit of the liver, including its oxygen gradient. Our liver-on-a-chip platform will deliver a microfluidic three-dimensional co-culture environment with stable synthetic and enzymatic function for at least 4 weeks. Sentinel cells that contain fluorescent biosensors will be integrated into the chip to provide multiplexed, real-time readouts of key liver functions and pathology. We are also developing a database to manage experimental data and harness external information to interpret the multimodal data and create a predictive platform.
doi:10.1186/scrt377
PMCID: PMC4028964  PMID: 24565476
4.  Determination and Extension of the Limits to Static Cold Storage with Subnormothermic Machine Perfusion 
Purpose
Static cold storage (SCS) of the liver for transplantation is limited by time. Continuation of metabolic activity leads to depletion of energy stores and loss of cellular function, which results in poor post-transplant function. Machine perfusion (MP) applied at the end of preservation may improve the viability of marginal liver grafts and provides information on the quality of the organ. We attempt to define the limits to SCS in terms of easily measurable perfusion parameters and investigate whether MP can improve liver viability.
Methods
Rat livers were cold-stored for 0, 24, 48, 72, and 120 hours, after which they were treated with subnormothermic machine perfusion (SNMP). Livers cold-stored for 48 and 72 hours were transplanted orthotopically with or without SNMP. During SNMP easily measurable parameters were monitored and adenosine triphosphate (ATP) content was measured following preservation and SNMP.
Results
ATP increased significantly during SNMP, but the recovered ATP content deteriorated with increased duration of SCS, with minimal improvement after 72 hours of SCS. Vascular resistance during SNMP increased with extended preservation. After 48 hours of SCS, orthotopic transplantation survival increased significantly from 50% to 100% with SNMP, but did not improve after 72 hours.
Conclusions
Vascular resistance and ATP recovery suggest a decrease in viability after 48 hours of SCS. Survival data confirms the loss of post-transplant graft function and supports the use of ATP and vascular resistance as useful indicators. Further, we show that the recoverability of a liver using SNMP is limited to 48 hours of SCS.
doi:10.5301/ijao.5000250
PMCID: PMC4091033  PMID: 24338652
Liver; Transplantation; Machine Perfusion; Preservation; Cold Ischemia; Adenosine Triphosphate
5.  Rat hepatocyte culture model of macrosteatosis: Effect of macrosteatosis induction and reversal on viability and liver-specific function 
Journal of hepatology  2013;59(6):1307-1314.
Background & Aims
A common cause of liver donor ineligibility is macrosteatosis. Recovery of such livers could enhance donor availability. Living donor studies have shown diet-induced reduction of macrosteatosis enables transplantation. However, cadaveric liver macrosteatotic reduction must be performed ex vivo within hours. Towards this goal, we investigated the effect of accelerated macrosteatosis reduction on hepatocyte viability and function using a novel system of macrosteatotic hepatocytes.
Methods
Hepatocytes isolated from lean Zucker rats were cultured in a collagen sandwich, incubated for 6 days in fatty acid-supplemented medium to induce steatosis, and then switched for 2 days to medium supplemented with lipid metabolism promoting agents. Intracellular lipid droplet size distribution and triglyceride, viability, albumin and urea secretion, and bile canalicular function were measured.
Results
Fatty acid-supplemented medium induced microsteatosis in 3 days and macrosteatosis in 6 days, the latter evidenced by large lipid droplets dislocating the nucleus to the cell periphery. Macrosteatosis significantly impaired all functions tested. Macrosteatosis decreased upon returning hepatocytes to standard medium, and the rate of decrease was 4-fold faster with supplemented agents, yielding 80% reduction in 2 days. Viability of macrosteatosis reduced hepatocytes was similar to control lean cells. Accelerated macrosteatotic reduction led to faster recovery of urea secretion and bile canalicular function, but not of albumin secretion.
Conclusions
Macrosteatosis reversibly decreases hepatocyte function and supplementary agents accelerate macrosteatosis reduction and some functional restoration with no effect on viability. This in vitro model may be useful to screen agents for macrosteatotic reduction in livers before transplantation.
doi:10.1016/j.jhep.2013.07.019
PMCID: PMC3899833  PMID: 23872604
Steatosis; Triglyceride; Albumin; Urea; Bile; Liver transplantation; Lipid metabolism
6.  SIMPLE MACHINE PERFUSION SIGNIFICANTLY ENHANCES HEPATOCYTE YIELDS OF ISCHEMIC AND FRESH RAT LIVERS 
Cell medicine  2013;4(3):109-123.
The scarcity of viable hepatocytes is a significant bottleneck in cell transplantation, drug discovery, toxicology, tissue engineering, and bioartificial assist devices, where trillions of high-functioning hepatocytes are needed annually. We took the novel approach of using machine perfusion to maximize cell recovery, specifically from uncontrolled cardiac death donors, the largest source of disqualified donor organs. In a rat model, we developed a simple 3 hour room temperature (20±2°C) machine perfusion protocol to treat non-premedicated livers exposed to 1 hour of warm (34°C) ischemia. Treated ischemic livers were compared to fresh, fresh-treated and untreated ischemic livers using viable hepatocyte yields and in vitro performance as quantitative endpoints. Perfusion treatment resulted in both a 25-fold increase in viable hepatocytes from ischemic livers, and a 40% increase from fresh livers. While cell morphology and function in suspension and plate cultures of untreated warm ischemic cells was significantly impaired, treated warm ischemic cells were indistinguishable from fresh hepatocytes. Further, a strong linear correlation between tissue ATP and cell yield enabled accurate evaluation of the extent of perfusion recovery. Maximal recovery of warm ischemic liver ATP content appears to be correlated with optimal flow through the microvasculature. These data demonstrate that the inclusion of a simple perfusion-preconditioning step can significantly increase the efficiency of functional hepatocyte yields and the number of donor livers that can be gainfully utilized.
doi:10.3727/215517912X658927
PMCID: PMC4243527  PMID: 25431743
DCD; NHBD; ischemia; ATP; cell isolation
7.  Perspectives on Non-Animal Alternatives for Assessing Sensitization Potential in Allergic Contact Dermatitis 
Skin sensitization remains a major environmental and occupational health hazard. Animal models have been used as the gold standard method of choice for estimating chemical sensitization potential. However, a growing international drive and consensus for minimizing animal usage have prompted the development of in vitro methods to assess chemical sensitivity. In this paper, we examine existing approaches including in silico models, cell and tissue based assays for distinguishing between sensitizers and irritants. The in silico approaches that have been discussed include Quantitative Structure Activity Relationships (QSAR) and QSAR based expert models that correlate chemical molecular structure with biological activity and mechanism based read-across models that incorporate compound electrophilicity. The cell and tissue based assays rely on an assortment of mono and co-culture cell systems in conjunction with 3D skin models. Given the complexity of allergen induced immune responses, and the limited ability of existing systems to capture the entire gamut of cellular and molecular events associated with these responses, we also introduce a microfabricated platform that can capture all the key steps involved in allergic contact sensitivity. Finally, we describe the development of an integrated testing strategy comprised of two or three tier systems for evaluating sensitization potential of chemicals.
doi:10.1007/s12195-011-0189-4
PMCID: PMC3985137  PMID: 24741377
Immunobiology; Skin sensitization; In silico approaches; QSAR; 2D cell based models; 3D skin tissue models; Integrated Testing Strategies; Microfabrication; Microfluidics
8.  Engineered Liver for Transplantation 
Current opinion in biotechnology  2013;24(5):893-899.
Orthotopic liver transplantation is the only definitive treatment for end stage liver failure and the shortage of donor organs severely limits the number of patients receiving transplants. Liver tissue engineering aims to address the donor liver shortage by creating functional tissue constructs to replace a damaged or failing liver. Despite decades of work, various bottoms-up, synthetic biomaterials approaches have failed to produce a functional construct suitable for transplantation. Recently, a new strategy has emerged using whole organ scaffolds as a vehicle for tissue engineering. This technique involves preparation of these organ scaffolds via perfusion decellularization with the resulting scaffold retaining the circulatory network of the native organ. This important phenomenon allows for the construct to be repopulated with cells and to be connected to the blood torrent upon transplantation. This opinion paper presents the current advances and discusses the challenges of creating fully functional transplantable liver grafts with this whole liver engineering approach.
doi:10.1016/j.copbio.2013.05.008
PMCID: PMC3783566  PMID: 23791465
9.  Microfluidic Enrichment of Mouse Epidermal Stem Cells and Validation of Stem Cell Proliferation In Vitro 
Tissue Engineering. Part C, Methods  2013;19(10):765-773.
Bulge stem cells reside in the lowest permanent portion of hair follicles and are responsible for the renewal of these follicles along with the repair of the epidermis during wound healing. These cells are identified by surface expression of CD34 and the α6-integrin. When CD34 and α6 double-positive cells are isolated and implanted into murine skin, they give rise to epidermis and hair follicle structures. The current gold standard for isolation of these stem cells is fluorescence-activated cell sorting (FACS) based on cell surface markers. Here, we describe an alternative method for CD34 bulge stem cell isolation: a microfluidic platform that captures stem cells based on cell surface markers. This method is relatively fast, requiring 30 min of time from direct introduction of murine skin tissue digestate into a two-stage microfluidic device to one-pass elution of CD34+ enriched cells with a purity of 55.8%±5.1%. The recovered cells remain viable and formed colonies with characteristic morphologies. When grown in culture, enriched cells contain a larger α6+ population than un-enriched cells.
doi:10.1089/ten.tec.2012.0638
PMCID: PMC3751333  PMID: 23394261
10.  Ex Vivo Gene Delivery to Hepatocytes: Techniques, Challenges, and Underlying Mechanisms 
Annals of biomedical engineering  2012;40(9):1851-1861.
Gene delivery to primary hepatocytes is an important tool for a number of applications including the study of liver cell biology and pathology, drug screening, and gene therapy. Robust transfection of primary hepatocytes, however, is significantly more difficult to achieve than in cell lines or readily dividing primary cells. In this report, we investigated in vitro gene delivery to both primary rat hepatocytes and Huh7.5.1 cells (a hepatoma cell line) using a number of viral and non-viral methods, including Lipofectamine 2000, FuGene HD, Nucleofection, Magnetofection, and lentiviruses. Our results showed that Lipofectamine 2000 is the most efficient reagent for green fluorescent protein (GFP) gene delivery to primary rat hepatocytes (33.3 ± 1.8% transfection efficiency) with minimal adverse effect on several hepatic functions, such as urea and albumin secretion. The lentiviral vectors used in this study exhibited undetectable gene delivery to primary rat hepatocytes but significant delivery to Huh7.5.1 cells (>80% transfection efficiency). In addition, we demonstrated lentiviral-based and spatially defined delivery of the GFP gene to Huh7.5.1 cells for use in biological microelectromechanical systems.
doi:10.1007/s10439-012-0555-y
PMCID: PMC3901163  PMID: 22484829
Non-viral transfection; Lentiviruses; Primary rat hepatocytes; Huh7.5.1; Transfection efficiency; Hepatic function
11.  Brain-on-a-chip microsystem for investigating traumatic brain injury: Axon diameter and mitochondrial membrane changes play a significant role in axonal response to strain injuries 
Technology  2014;2(2):106-.
Diffuse axonal injury (DAI) is a devastating consequence of traumatic brain injury, resulting in significant axon and neuronal degeneration. Currently, therapeutic options are limited. Using our brain-on-a-chip device, we evaluated axonal responses to DAI. We observed that axonal diameter plays a significant role in response to strain injury, which correlated to delayed elasticity and inversely correlated to axonal beading and axonal degeneration. When changes in mitochondrial membrane potential (MMP) were monitored an applied strain injury threshold was noted, below which delayed hyperpolarization was observed and above which immediate depolarization occurred. When the NHE-1 inhibitor EIPA was administered before injury, inhibition in both hyperpolarization and depolarization occurred along with axonal degeneration. Therefore, axonal diameter plays a significant role in strain injury and our brain-on-a-chip technology can be used both to understand the biochemical consequences of DAI and screen for potential therapeutic agents.
doi:10.1142/S2339547814500095
PMCID: PMC4120884  PMID: 25101309
12.  Eradication of multidrug-resistant A. baumannii in burn wounds by antiseptic pulsed electric field 
Technology  2014;2(2):153-160.
Emerging bacterial resistance to multiple drugs is an increasing problem in burn wound management. New non-pharmacologic interventions are needed for burn wound disinfection. Here we report on a novel physical method for disinfection: antiseptic pulsed electric field (PEF) applied externally to the infected burns. In a mice model, we show that PEF can reduce the load of multidrug resistant Acinetobacter baumannii present in a full thickness burn wound by more than four orders of magnitude, as detected by bioluminescence imaging. Furthermore, using a finite element numerical model, we demonstrate that PEF provides non-thermal, homogeneous, full thickness treatment for the burn wound, thus, overcoming the limitation of treatment depth for many topical antimicrobials. These modeling tools and our in vivo results will be extremely useful for further translation of the PEF technology to the clinical setting, as they provide the essential elements for planning of electrode design and treatment protocol.
PMCID: PMC4117356  PMID: 25089285
13.  Reprogramming of Intestinal Glucose Metabolism and Glycemic Control in Rats After Gastric Bypass 
Science (New York, N.Y.)  2013;341(6144):406-410.
The resolution of type 2 diabetes after Roux-en-Y gastric bypass (RYGB) attests to the important role of the gastrointestinal tract in glucose homeostasis. Previous studies in RYGB-treated rats have shown that the Roux limb displays hyperplasia and hypertrophy. Here, we report that the Roux limb of RYGB-treated rats exhibits reprogramming of intestinal glucose metabolism to meet its increased bioenergetic demands; glucose transporter-1 is up-regulated, basolateral glucose uptake is enhanced, aerobic glycolysis is augmented, and glucose is directed toward metabolic pathways that support tissue growth. We show that reprogramming of intestinal glucose metabolism is triggered by the exposure of the Roux limb to undigested nutrients. We demonstrate by positron emission tomography–computed tomography scanning and biodistribution analysis using 2-deoxy-2-[18F]fluoro-D-glucose that reprogramming of intestinal glucose metabolism renders the intestine a major tissue for glucose disposal, contributing to the improvement in glycemic control after RYGB.
doi:10.1126/science.1235103
PMCID: PMC4068965  PMID: 23888041
14.  Organomatics and organometrics: Novel platforms for long-term whole-organ culture 
Technology  2014;2(1):13-.
Organ culture systems are instrumental as experimental whole-organ models of physiology and disease, as well as preservation modalities facilitating organ replacement therapies such as transplantation. Nevertheless, a coordinated system of machine perfusion components and integrated regulatory control has yet to be fully developed to achieve long-term maintenance of organ function ex vivo. Here we outline current strategies for organ culture, or organomatics, and how these systems can be regulated by means of computational algorithms, or organometrics, to achieve the organ culture platforms anticipated in modern-day biomedicine.
doi:10.1142/S2339547814300029
PMCID: PMC4097862  PMID: 25035864
15.  Non-thermal, pulsed electric field cell ablation: A novel tool for regenerative medicine and scarless skin regeneration 
Technology  2013;1(1):1-8.
High voltage, short pulsed electric fields (PEF) is a non-thermal ablation method, in which defined PEF irreversibly destabilize cell membranes, while preserving other tissue components such as the extracellular matrix (ECM). In the present report, we show that PEF ablated rat skin retains its microvascular blood supply and ECM structure. Complete regeneration of epidermis, hair follicles, sebaceous glands, and the panniculus carnosusis observed two months after the ablation. Our results clearly indicate that non-thermal PEF has the potential to be a powerful and novel tool for scarless tissue regeneration.
doi:10.1142/S233954781320001X
PMCID: PMC4078877  PMID: 24999487
16.  Stem Cells for Liver Repopulation 
Purpose of review
The capacity of the liver to regenerate and maintain a constant size despite injury is unique. However, the exact mechanisms are not completely clear. Cell transplantation has been proposed as an alternative treatment of liver diseases. Recent progress has been reported on the generation of stem/progenitor cells that may differentiate towards the hepatic lineage. However, it is currently difficult to determine which of the stem/progenitor cell populations are the best for therapy of a given disease.
Recent findings
The limited access to donor human hepatocytes has opened a great interest on the generation of hepatocyte-like cells. Several potential cell sources have been identified. However, general standardization of the methods to evaluate these cells is particularly important for the promise of stem/progenitor-derived hepatocyte-based therapies. Moreover, innovations aimed at improving hepatocyte delivery, survival and engraftment have recently opened the field of organ engineering that may improve the perspective of liver repopulation.
Summary
Here we review current evidence reported from the perspective of potential clinical applications of different hepatic cell sources with repopulation capacities and the future perspectives and tools that can facilitate the translation of laboratory work into clinical success.
doi:10.1097/MOT.0b013e3283328070
PMCID: PMC3725723  PMID: 19779345
Hepatocyte transplantation; xenogenic hepatocytes; stem cell-derived hepatocytes; liver tissue engineering
17.  Target DNA detection and quantitation on a single cell with single base resolution 
Technology  2013;1(1):88-.
In this report, we present a new method for sensitive detection of short DNA sites in single cells with single base resolution. The method combines peptide nucleic acid (PNA) openers as the tagging probes, together with isothermal rolling circle amplification (RCA) and fluorescence-based detection, all performed in a cells-in-flow format. Bis-PNAs provide single base resolution, while RCA ensures linear signal amplification. We applied this method to detect the oncoviral DNA inserts in cancer cell lines using a flow-cytometry system. We also demonstrated quantitative detection of the selected signature sites within single cells in microfluidic nano-liter droplets. Our results show single-nucleotide polymorphism (SNP) discrimination and detection of copy-number variations (CNV) under isothermal non-denaturing conditions. This new method is ideal for many applications in which ultra-sensitive DNA characterization with single base resolution is desired on the level of single cells.
doi:10.1142/S2339547813500088
PMCID: PMC4073798  PMID: 24977169
18.  A novel ultrathin collagen nanolayer assembly for 3-D microtissue engineering: Layer-by-layer collagen deposition for long-term stable microfluidic hepatocyte culture 
Technology  2014;2(1):67-74.
The creation of stable hepatocyte cultures using cell-matrix interactions has proven difficult in microdevices due to dimensional constraints limiting the utility of classic tissue culture techniques that involve the use of hydrogels such as the collagen “double gel” or “overlay”. To translate the collagen overlay technique into microdevices, we modified collagen using succinylation and methylation reactions to create polyanionic and polycationic collagen solutions, and deposited them layer-by-layer to create ultrathin collagen nanolayers on hepatocytes. These ultrathin collagen layers covered hepatocytes in microdevices and 1) maintained cell morphology, viability, and polarity, 2) induced bile canalicular formation and actin reorganization, and 3) maintained albumin and urea secretions and CYP activity similar to those observed in hepatocytes in collagen double gel hepatocytes in plate cultures. Beyond the immediate applications of this technique to create stable, in vitro microfluidic hepatocyte cultures for drug toxicity testing, this technique is generally applicable as a thin biomaterial for other 3D microtissues.
doi:10.1142/S2339547814500083
PMCID: PMC4054686  PMID: 24932459
19.  PLGA-polymer encapsulating tumor antigen and CpG DNA administered into the tumor microenvironment elicits a systemic antigen-specific IFN-γ response and enhances survival 
Journal of cancer therapy  2013;4(1):280-290.
Critical to the generation of an effective therapeutic antitumor immune response is the elicitation of effective antigen presentation coupled with overcoming tumor-immune escape mechanisms. Towards this end, we aimed to understand the therapeutic effectiveness of a polymer based vaccine approach at enhancing the anti-tumor responses in a tumor-bearing mouse model. While we and others have previously demonstrated the effectiveness of PLGA based systems in delivering antigen etc., studies scarcely focus on understanding the immunological mechanisms of polymer based therapies in tumor bearing treatment models. Considering tumors modulate the immune system and consequently the efficacy of therapies, understanding treatment mechanisms in the presence of tumor will help lead to more efficacious treatment options. We demonstrate here that a poly(lactic-co-glycolic acid) (PLGA) based delivery system encapsulating tumor antigen (OVA) and the TLR9 agonist CpG motif DNA administered into the tumor microenvironment initiates an effective type 1 mediated (IFN-γ producing) anti-tumor response in a syngeneic murine model of T cell lymphoma (E.G7-OVA). Although E.G7-OVA tumors spontaneously generate antigen specific CTLs in draining lymph nodes (LN), tumors progress rapidly. Modulation of the tumor microenvironment via local PLGA based therapy led to the generation of a systemic antigen specific Th1 response, absent in the non-polymer delivery method, subsequently associated with reduced tumor growth and prolongation of survival. These studies provide further insight into the use of a PLGA-based therapeutic approach at modulating the tumor microenvironment and highlight the need for analyzing the treatment effects in a tumor bearing model.
doi:10.4236/jct.2013.41035
PMCID: PMC3670804  PMID: 23741626
Cytokine; Immune response; Immunomodulation; Macrophage; Microencapsulation; Vaccine
20.  ANALYSIS OF DENDRITIC CELL STIMULATION UTILIZING A MULTI-FACETED NANOPOLYMER DELIVERY SYSTEM AND THE IMMUNE MODULATOR 1-METHYL TRYPTOPHAN 
Nano LIFE  2010;1(3-4):239-250.
Dendritic cells (DCs) play a pivotal role in immune modulation. Therefore, understanding and regulating the mechanism of DC activation is paramount for functional optimization of any immunotherapy strategy. In particular, the paradoxical ability of DCs to secrete the immune suppressive enzyme indoleamine 2, 3-dioxygenase (IDO) and the suppressive cytokine IL-10 during the course of, and in response to, stimulation is of great interest. 1-Methyl-Tryptophan (1 MT) is a known inhibitor of IDO and has thus been administered in numerous in vitro and in vivo systems to block IDO activity. However, the effect 1 MT has on DCs beyond inhibiting IDO, especially in therapeutic models, has rarely been analyzed. In the current study, we have administered 1 MT via a nanopolymer-based delivery system in conjunction with an antigen (ovalbumin, OVA) and an adjuvant (CpG motif DNA) to determine both the effects of 1 MT on DCs and the resulting efficacy of the polymer-based treatments. 1 MT delivery alone, either via the polymer-based delivery vehicle or dissolved in solution, induced no significant change in DC activation as measured by surface expression of CD80, CD86, and MHCII and several secreted products such as IL-12. These same factors were upregulated however, when 1 MT was delivered in conjunction with OVA and CpG. Although soluble delivery of these components increased the levels of expression and secretion of key proteins, a differential effect of DC stimulation was seen as a result of the polymer delivery system. The T cell suppressive IL-10 secretion was lower with the polymer-based treatments and IL-12 immune-enhancing secretion was increased when 1 MT was supplemented into the polymer system. As a result, including 1 MT in the polymers along with OVA and CpG was seen to have additional effects on DC stimulation and was able to shift DCs to a state more indicative of inducing a Th1-type response.
doi:10.1142/S1793984410000171
PMCID: PMC3998209  PMID: 24772192
Dendritic cells; DC; PLGA; 1-methyl-tryptophan; 1MT
21.  Secreted Factors from Bone Marrow Stromal Cells Upregulate IL-10 and Reverse Acute Kidney Injury 
Stem Cells International  2012;2012:392050.
Acute kidney injury is a devastating syndrome that afflicts over 2,000,000 people in the US per year, with an associated mortality of greater than 70% in severe cases. Unfortunately, standard-of-care treatments are not sufficient for modifying the course of disease. Many groups have explored the use of bone marrow stromal cells (BMSCs) for the treatment of AKI because BMSCs have been shown to possess unique anti-inflammatory, cytoprotective, and regenerative properties in vitro and in vivo. It is yet unresolved whether the primary mechanisms controlling BMSC therapy in AKI depend on direct cell infusion, or whether BMSC-secreted factors alone are sufficient for mitigating the injury. Here we show that BMSC-secreted factors are capable of providing a survival benefit to rats subjected to cisplatin-induced AKI. We observed that when BMSC-conditioned medium (BMSC-CM) is administered intravenously, it prevents tubular apoptosis and necrosis and ameliorates AKI. In addition, we observed that BMSC-CM causes IL-10 upregulation in treated animals, which is important to animal survival and protection of the kidney. In all, these results demonstrate that BMSC-secreted factors are capable of providing support without cell transplantation, and the IL-10 increase seen in BMSC-CM-treated animals correlates with attenuation of severe AKI.
doi:10.1155/2012/392050
PMCID: PMC3539665  PMID: 23319959
22.  Optimality and thermodynamics determine the evolution of transcriptional regulatory networks† 
Molecular bioSystems  2011;8(2):511-530.
Transcriptional motifs are small regulatory interaction patterns that regulate biological functions in highly-interacting cellular networks. Recently, attempts have been made to explain the significance of transcriptional motifs through dynamic function. However, fundamental questions remain unanswered. Why are certain transcriptional motifs with similar dynamic function abundant while others occur rarely? What are the criteria for topological generalization of these motifs into complex networks? Here, we present a novel paradigm that combines non-equilibrium thermodynamics with multiobjective-optimality for network analysis. We found that energetic cost, defined herein as specific dissipation energy, is minimal at the optimal environmental conditions and it correlates inversely with the abundance of the network motifs obtained experimentally for E. coli and S. cerevisiae. This yields evidence that dissipative energetics is the underlying criteria used during evolution for motif selection and that biological systems during transcription tend towards evolutionary selection of subgraphs which produces minimum specific heat dissipation under optimal conditions, thereby explaining the abundance/rare occurrence of some motifs. We show that although certain motifs had similar dynamical functionality, they had significantly different energetic cost, thus explaining the abundance/rare occurrence of these motifs. The presented insights may establish global thermodynamic analysis as a backbone in designing and understanding complex networks systems, such as metabolic and protein interaction networks.
doi:10.1039/c1mb05177f
PMCID: PMC3964374  PMID: 22076617
23.  Pharmacokinetics of Natural and Engineered Secreted Factors Delivered by Mesenchymal Stromal Cells 
PLoS ONE  2014;9(2):e89882.
Transient cell therapy is an emerging drug class that requires new approaches for pharmacological monitoring during use. Human mesenchymal stem cells (MSCs) are a clinically-tested transient cell therapeutic that naturally secrete anti-inflammatory factors to attenuate immune-mediated diseases. MSCs were used as a proof-of-concept with the hypothesis that measuring the release of secreted factors after cell transplantation, rather than the biodistribution of the cells alone, would be an alternative monitoring tool to understand the exposure of a subject to MSCs. By comparing cellular engraftment and the associated serum concentration of secreted factors released from the graft, we observed clear differences between the pharmacokinetics of MSCs and their secreted factors. Exploration of the effects of natural or engineered secreted proteins, active cellular secretion pathways, and clearance mechanisms revealed novel aspects that affect the systemic exposure of the host to secreted factors from a cellular therapeutic. We assert that a combined consideration of cell delivery strategies and molecular pharmacokinetics can provide a more predictive model for outcomes of MSC transplantation and potentially other transient cell therapeutics.
doi:10.1371/journal.pone.0089882
PMCID: PMC3931832  PMID: 24587097
24.  Isolation Rearing Impairs Wound Healing and is Associated with Increased Locomotion and Decreased Immediate Early Gene Expression in the Medial Prefrontal Cortex of Juvenile Rats 
Neuroscience  2007;151(2):589-603.
In addition to its maladaptive effects on psychiatric function, psychosocial deprivation impairs recovery from physical illness. Previously, we found that psychosocial deprivation, modeled by isolation rearing, depressed immediate early gene (IEG) expression in the medial prefrontal cortex (mPFC) and increased locomotion in the open field test (Levine, Youngs et al. 2007). In the present study, we examined whether similar changes in behavior and gene expression are associated with the maladaptive effects of psychosocial deprivation on physical injury healing. After weaning, anesthetized rats were subjected to a 20% total body surface area third degree burn injury and were subsequently either group or isolation reared. After four weeks of either isolation or group rearing (a period that encompasses rodent childhood and early adolescence), rats were sacrificed, and their healing and gene expression in the mPFC were assessed. Locomotion in the open field test was examined at 3 weeks post burn injury. We found that: 1) gross wound healing was significantly impaired in isolation reared rats compared to group reared rats, 2) locomotion was increased and IEG expression was suppressed for isolation reared rats during burn injury healing, 3) the decreased activity in the open field and increased IEG expression was greater for burn injury healing group reared rats than for uninjured group reared rats, 4) the degree of hyperactivity and IEG suppression was relatively similar between isolation reared rats during burn injury compared to uninjured isolation reared rats, 5) behavioral hyperactivity to novelty (the open field test) along with IEG suppression may constitute a detectable biomarker of isolation rearing during traumatic physical injury. Implications of the findings for understanding, assessing, and treating the maladaptive effects of psychosocial deprivation on physical healing during childhood are discussed.
doi:10.1016/j.neuroscience.2007.10.014
PMCID: PMC3923447  PMID: 18063315
25.  AN ORGANOTYPIC UNIAXIAL STRAIN MODEL USING MICROFLUIDICS 
Lab on a chip  2013;13(3):10.1039/c2lc41063j.
Traumatic brain injuries are the leading cause of disability each year in the US. The most common and devastating consequence is the stretching of axons caused by shear deformation that occurs during rotational acceleration of the brain during injury. The injury effects on axonal molecular and functional events are not fully characterized. We have developed a strain injury model that maintains the three dimensional cell architecture and neuronal networks found in vivo with the ability to visualize individual axons and their response to a mechanical injury. The advantage of this model is that it can apply uniaxial strains to axons that make functional connections between two organotypic slices and injury responses can be observed in real-time and over long term. This uniaxial strain model was designed to be capable of applying an array of mechanical strains at various rates of strain, thus replicating a range of modes of axonal injury. Long term culture, preservation of slice and cell orientation, and slice-slice connection on the device was demonstrated. The device has the ability to strain either individual axons or bundles of axons through the control of microchannel dimensions. The fidelity of the model was verified by observing characteristic responses to various strain injuries which included axonal beading, delayed elastic effects and breakdown in microtubules. Microtubule breakdown was shown to be dependent on the degree of the applied strain field, where maximal breakdown was observed at peak strain and minimal breakdown is observed at low strain. This strain injury model could be a powerful tool in assessing strain injury effects on functional axonal connections.
doi:10.1039/c2lc41063j
PMCID: PMC3546521  PMID: 23233120

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