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In vitro studies of drug toxicity and drug-drug interactions are crucial for drug development efforts. Currently, the utilization of primary human hepatocytes (PHHs) is the de facto standard for this purpose, due to their functional xenobiotic response and drug metabolizing CYP450 enzyme metabolism. However, PHHs are scarce, expensive, require laborious maintenance, and exhibit lot-to-lot heterogeneity. Alternative human in vitro platforms include hepatic cell lines, which are easy to access and maintain, and induced pluripotent stem cell (iPSC) derived hepatocytes. In this study, we provide a direct comparison of drug induced CYP3A4 and PXR expression levels of PHHs, hepatic cell lines Huh7 and HepG2, and iPSC derived hepatocyte like cells. Confluently cultured Huh7s exhibited an improved CYP3A4 expression and were inducible by up to 4.9-fold, and hepatocytes differentiated from human iPSCs displayed a 3.3-fold CYP3A4 induction. In addition, an increase in PXR expression levels was observed in both hepatic cell lines and iPSC derived hepatocytes upon rifampicin treatment, whereas a reproducible increase in PXR expression was not achieved in PHHs. Our results indicate that both hepatoma originated cell lines and iPSCs may provide alternative sources to primary hepatocytes, providing reliable and reproducible results for CYP3A4/PXR metabolism, upon in vitro maturation. This study may serve as a guide for the selection of suitable and feasible in vitro platforms for drug-drug interaction and toxicology studies.

Gaurav Sharma1, Kaushal Rege2,3, David E Budil4, Martin L Yarmush2,5, Constantinos Mavroidis11Department of Mechanical and Industrial Engineering; 4Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, USA; 2The Center for Engineering in Medicine (CEM), Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA; 3Department of Chemical Engineering, Arizona State University, Tempe, AZ, USA; 5Department of Biomedical Engineering, Rutgers University, NJ, USAAbstract: We describe the molecular dynamics (MD)-aided engineering design of mutant peptides based on the α-helical coiled-coil GCN4 leucine zipper peptide (GCN4-p1) in order to obtain environmentally-responsive nanotweezers. The actuation mechanism of the nanotweezers depends on the modification of electrostatic charges on the residues along the length of the coiled coil. Modulating the solution pH between neutral and acidic values results in the reversible movement of helices toward and away from each other and creates a complete closed-open-closed transition cycle between the helices. Our results indicate that the mutants show a reversible opening of up to 15 Å (1.5 nm; approximately 150% of the initial separation) upon pH actuation. Investigation on the physicochemical phenomena that influence conformational properties, structural stability, and reversibility of the coiled-coil peptide-based nanotweezers revealed that a rationale- and design-based approach is needed to engineer stable peptide or macromolecules into stimuli-responsive devices. The efficacy of the mutant that demonstrated the most significant reversible actuation for environmentally responsive modulation of DNA-binding activity was also demonstrated. Our results have significant implications in bioseparations and in the engineering of novel transcription factors.Keywords: bionanotechnology, nanotweezers, coiled-coil, GCN4, leucine zipper, molecular dynamics, environmentally responsive peptides, transcription factor engineering

Abstract Red blood cell (RBC) transfusions facilitate many life-saving acute and chronic interventions. Transfusions are enabled through the gold-standard hypothermic storage of RBCs. Today, the demand for RBC units is unfulfilled, partially due to the limited storage time, 6 weeks, in hypothermic storage. This time limit stems from high metabolism-driven storage lesions at +1-6 °C. A recent and promising alternative to hypothermic storage is the supercooled storage of RBCs at subzero temperatures, pioneered by our group. Here, we report on long-term supercooled storage of human RBCs at physiological hematocrit levels for up to 23 weeks. Specifically, we assess hypothermic RBC additive solutions for their ability to sustain supercooled storage. We find that a commercially formulated next-generation solution (Erythro-Sol 5) enables the best storage performance and can form the basis for further improvements to supercooled storage. Our analyses indicate that oxidative stress is a prominent time- and temperature-dependent injury during supercooled storage. Thus, we report on improved supercooled storage of RBCs at −5 °C by supplementing Erythro-Sol 5 with the exogenous antioxidants, resveratrol, serotonin, melatonin, and Trolox. Overall, this study shows the long-term preservation potential of supercooled storage of RBCs and establishes a foundation for further improvement toward clinical translation.

In severe burn injury the unique setting of a depleted, dysfunctional immune system along with a loss of barrier function commonly results in opportunistic infections that eventually proof fatal. Unfortunately, the dynamic sequence of bacterial contamination, colonization and eventually septic invasion with bacteria such as Pseudomonas species is still poorly understood although a limiting factor in clinical decision making. Increasing evidence supports the notion that inhibition of bacterial translocation into the wound site may be an effective alternative to prevent infection. In this context we investigated the role of the mammalian Chitinase-3-Like-1 (CHI3L1) non-enyzmatic protein predominately expressed on epithelial as well as innate immune cells as a potential bacterial-translocation-mediating factor. We show a strong trend that a modulation of chitinase expression is likely to be effective in reducing mortality rates in a mouse model of burn injury with superinfection with the opportunistic PA14 Pseudomonas strain, thus demonstrating possible clinical leverage.

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.

We report an all-in-one platform - ScanDrop - for the rapid and specific capture, detection, and identification of bacteria in drinking water. The ScanDrop platform integrates droplet microfluidics, a portable imaging system, and cloud-based control software and data storage. The cloud-based control software and data storage enables robotic image acquisition, remote image processing, and rapid data sharing. These features form a "cloud" network for water quality monitoring. We have demonstrated the capability of ScanDrop to perform water quality monitoring via the detection of an indicator coliform bacterium, Escherichia coli, in drinking water contaminated with feces. Magnetic beads conjugated with antibodies to E. coli antigen were used to selectively capture and isolate specific bacteria from water samples. The bead-captured bacteria were co-encapsulated in pico-liter droplets with fluorescently-labeled anti-E. coli antibodies, and imaged with an automated custom designed fluorescence microscope. The entire water quality diagnostic process required 8 hours from sample collection to online-accessible results compared with 2-4 days for other currently available standard detection methods.

Supercooled preservation (SCP) is a technology that involves cooling a substance below its freezing point without initiating ice crystal formation. It is a promising alternative to prolong the preservation time of cells, tissues, engineered tissue products, and organs compared to the current practices of hypothermic storage. Two-dimensional (2D) engineered tissues are extensively used in in vitro research for drug screening and development and investigation of disease progression. Despite their widespread application, there is a lack of research on the SCP of 2D-engineered tissues. In this study, we presented the effects of SCP at −2 and −6°C on primary rat hepatocyte (PRH) monolayers for the first time and compared cell viability and functionality with cold storage (CS, + 4°C). We preserved PRH monolayers in two different commercially available solutions: Hypothermosol-FRS (HTS-FRS) and the University of Wisconsin (UW) with and without supplements (i.e., polyethylene glycol (PEG) and 3-O-Methyl-Α-D-Glucopyranose (3-OMG)). Our findings revealed that UW with and without supplements were inadequate for the short-term preservation of PRH monolayers for both SCP and CS with high viability, functionality, and monolayer integrity. The combination of supplements (PEG and 3-OMG) in the HTS-FRS solution outperformed the other groups and yielded the highest viability and functional capacity. Notably, PRH monolayers exhibited superior viability and functionality when stored at −2°C through SCP for up to 3 days compared to CS. Overall, our results demonstrated that SCP is a feasible approach to improving the short-term preservation of PRH monolayers and enables readily available 2D-engineered tissues to advance in vitro research. Furthermore, our findings provide insights into preservation outcomes across various biological levels, from cells to tissues and organs, contributing to the advancement of bioengineering and biotechnology.

Normothermic machine perfusion has previously been demonstrated to restore damaged warm ischemic livers to transplantable condition in animal models. However, the mechanisms of recovery are unclear, preventing rational optimization of perfusion systems and slowing clinical translation of machine perfusion. In this study, organ recovery time and major perfusate shortcomings were evaluated using a comprehensive metabolic analysis of organ function in perfusion prior to successful transplantation. Two groups, Fresh livers and livers subjected to 1 hr of warm ischemia (WI) received perfusion for a total preservation time of 6 hrs, followed by successful transplantation. 24 metabolic fluxes were directly measured and 38 stoichiometrically-related fluxes were estimated via a mass balance model of the major pathways of energy metabolism. This analysis revealed stable metabolism in Fresh livers throughout perfusion while identifying two distinct metabolic states in WI livers, separated at t = 2 hrs, coinciding with recovery of oxygen uptake rates to Fresh liver values. This finding strongly suggests successful organ resuscitation within 2 hrs of perfusion. Overall perfused livers regulated metabolism of perfusate substrates according to their metabolic needs, despite supraphysiological levels of some metabolites. This study establishes the first integrative metabolic basis for the dynamics of recovery during perfusion treatment of marginal livers. Our initial findings support enhanced oxygen delivery for both timely recovery and long-term sustenance. These results are expected to lead the optimization of the treatment protocols and perfusion media from a metabolic perspective, facilitating translation to clinical use.

Supercooling preservation holds the potential to drastically extend the preservation time of organs, tissues and engineered tissue products, and fragile cell types that do not lend themselves well to cryopreservation or vitrification. Here, we investigate the effects of supercooling preservation (SCP at -4(o)C) on primary rat hepatocytes stored in cryovials and compare its success (high viability and good functional characteristics) to that of static cold storage (CS at +4(o)C) and cryopreservation. We consider two prominent preservation solutions a) Hypothermosol (HTS-FRS) and b) University of Wisconsin solution (UW) and a range of preservation temperatures (-4 to -10 (o)C). We find that there exists an optimum temperature (-4(o)C) for SCP of rat hepatocytes which yields the highest viability; at this temperature HTS-FRS significantly outperforms UW solution in terms of viability and functional characteristics (secretions and enzymatic activity in suspension and plate culture). With the HTS-FRS solution we show that the cells can be stored for up to a week with high viability (~56%); moreover we also show that the preservation can be performed in large batches (50 million cells) with equal or better viability and no loss of functionality as compared to smaller batches (1.5 million cells) performed in cryovials.

Hair cycling is a prime example of stem cell dependent tissue regeneration and replenishment, and its regulatory mechanisms remain poorly understood. In the present study, we evaluated the effect of a blockage in terminal keratinocytic lineage differentiation in the Foxn1(-/-) nude phenotype on the epithelial progeny. Most notably we found a constitutive upregulation of LIM homeobox protein 2 (Lhx2), a marker gene of epithelial stem cellness indispensible for hair cycle progression. However, histological evidence along with an erratic, acyclic rise of otherwise suppressed CyclinD1 levels along with several key markers of keratinocyte lineage differentiation indicate a frustrated expansion of epithelial stem cell niches in skin. In addition, CD49f/CD34/CD200-based profiling demonstrated highly significant shifts in subpopulations of epithelial progeny. Intriguingly this appeared to include the expansion of Oct4+ stem cells in dermal fractions of skin isolates in the Foxn1 knock-out opposed to wild type. Overall our findings indicate that the Foxn1(-/-) phenotype has a strong impact on epithelial progeny and thus offers a promising model to study maintenance and regulation of stem cell niches within skin not feasible in other in vitro or in vivo models.

The abundant flavonoid aglycone, naringenin, which is responsible for the bitter taste in grapefruits, has been shown to possess hypolipidemic and anti-inflammatory effects both in vitro and in vivo. Recently, our group demonstrated that naringenin inhibits hepatitis C virus (HCV) production, while others demonstrated its potential in the treatment of hyperlipidemia and diabetes. However, naringenin suffers from low oral bioavailability critically limiting its clinical potential. In this study, we demonstrate that the solubility of naringenin is enhanced by complexation with β-cyclodextrin, an FDA approved excipient. Hydroxypropoyl-β-cyclodextrin (HPβCD), specifically, increased the solubility of naringenin by over 400-fold, and its transport across a Caco-2 model of the gut epithelium by 11-fold. Complexation of naringenin with HPβCD increased its plasma concentrations when fed to rats, with AUC values increasing by 7.4-fold and C(max) increasing 14.6-fold. Moreover, when the complex was administered just prior to a meal it decreased VLDL levels by 42% and increased the rate of glucose clearance by 64% compared to naringenin alone. These effects correlated with increased expression of the PPAR co-activator, PGC1α in both liver and skeletal muscle. Histology and blood chemistry analysis indicated this route of administration was not associated with damage to the intestine, kidney, or liver. These results suggest that the complexation of naringenin with HPβCD is a viable option for the oral delivery of naringenin as a therapeutic entity with applications in the treatment of dyslipidemia, diabetes, and HCV infection.

With over 110,000 patients waiting for organ transplantation, the current crisis in organ transplantation is based on a lack of donors after brain-death (DBD). A very large alternative pool of donor organs that remain untapped are the donors after cardiac death (DCD), recovered after cardiac activity has ceased and therefore sustained some ischemic injury. Machine perfusion has been proposed as a novel modality of organ preservation and treatment to render such cadaveric organs, and in particular livers, transplantable. Two key issues that remain unaddressed are how to assess whether a DCD liver is damaged beyond repair, and whether machine perfusion has rendered an injured organ sufficiently viable for transplantation. In this work, we present a metabolic analysis of the transient responses of cadaveric rat livers during normothermic machine perfusion (NMP), and develop an index of ischemia that enables evaluation of the organ ischemic injury level. Further, we perform a discriminant analysis to construct a classification algorithm with >0.98 specificity to identify whether a given perfused liver is ischemic or fresh, in effect a precursor for an index of transplantability and a basis for the use of statistical process control measures for automated feedback control of treatment of ischemic injury in DCD livers. The analyses yield an index based on squared prediction error (SPE) as log(SPE) >1.35 indicating ischemia. The differences between metabolic functions of fresh and ischemic livers during perfusion are outlined and the metabolites that varied significantly for ischemic livers are identified as ornithine, arginine, albumin and tyrosine.

The elucidation of the effect of extracellular matrices on hepatocellular metabolism is critical to understand the mechanism of functional upregulation. We have developed a system using natural extracellular matrices [Adipogel] for enhanced albumin synthesis of rat hepatocyte cultures for a period of 10 days as compared to collagen sandwich cultures. Primary rat hepatocytes isolated from livers of female Lewis rats recover within 4 days of culture from isolation induced injury while function is stabilized at 7 days post-isolation. Thus, the culture period can be classified into three distinct stages viz. recovery stage [day 0-4], pre-stable stage [day 5-7] and the stable stage [day 8-10]. A Metabolic Flux Analysis of primary rat hepatocytes cultured in Adipogel was performed to identify the key metabolic pathways modulated as compared to collagen sandwich cultures. In the recovery stage [day 4], the collagen-soluble Adipogel cultures shows an increase in TriCarboxylic Acid [TCA] cycle fluxes; in the pre-stable stage [day 7], there is an increase in PPP and TCA cycle fluxes while in the stable stage [day 10], there is a significant increase in TCA cycle, urea cycle fluxes and amino acid uptake rates concomitant with increased albumin synthesis rate as compared to collagen sandwich cultures throughout the culture period. Metabolic analysis of the collagen-soluble Adipogel condition reveals significantly higher transamination reaction fluxes, amino acid uptake and albumin synthesis rates for the stable vs. recovery stages of culture. The identification of metabolic pathways modulated for hepatocyte cultures in presence of Adipogel will be a useful step to develop an optimization algorithm to further improve hepatocyte function for Bioartificial Liver Devices. The development of this framework for upregulating hepatocyte function in Bioartificial Liver Devices will facilitate the utilization of an integrated experimental and computational approach for broader applications of Adipogel in tissue e engineering and regenerative medicine.

Endothelial cells represent an important barrier between the intravascular compartment and extravascular tissues, and therefore serve as key sensors, communicators, and amplifiers of danger signals in innate immunity and inflammation. Double stranded DNA (dsDNA) released from damaged host cells during injury or introduced by pathogens during infection, has emerged as a potent danger signal. While the dsDNA-mediated immune response has been extensively studied in immune cells, little is known about the direct and indirect effects of dsDNA on the vascular endothelium. In this study we show that direct dsDNA stimulation of endothelial cells induces a potent proinflammatory response as demonstrated by increased expression of ICAM1, E-selectin and VCAM1, and enhanced leukocyte adhesion. This response was dependent on the stress kinases JNK and p38 MAPK, required the activation of proinflammatory transcription factors NFκB and IRF3, and triggered the robust secretion of TNFα for sustained secondary activation of the endothelium. DNA-induced TNFα secretion proved to be essential in vivo, as mice deficient in the TNF receptor were unable to mount an acute inflammatory response to dsDNA. Our findings suggest that the endothelium plays an active role in mediating dsDNA-induced inflammatory responses, and implicate its importance in establishing an acute inflammatory response to sterile injury or systemic infection, where host or pathogen derived dsDNA may serve as a danger signal.

Disruption of lipid and carbohydrate homeostasis is an important factor in the development of prevalent metabolic diseases such as diabetes, obesity, and atherosclerosis. Therefore, small molecules that could reduce insulin dependence and regulate dyslipidemia could have a dramatic effect on public health. The grapefruit flavonoid naringenin has been shown to normalize lipids in diabetes and hypercholesterolemia, as well as inhibit the production of HCV. Here, we demonstrate that naringenin regulates the activity of nuclear receptors PPARalpha, PPARgamma, and LXRalpha. We show it activates the ligand-binding domain of both PPARalpha and PPARgamma, while inhibiting LXRalpha in GAL4-fusion reporters. Using TR-FRET, we show that naringenin is a partial agonist of LXRalpha, inhibiting its association with Trap220 co-activator in the presence of TO901317. In addition, naringenin induces the expression of PPARalpha co-activator, PGC1alpha. The flavonoid activates PPAR response element (PPRE) while suppressing LXRalpha response element (LXRE) in human hepatocytes, translating into the induction of PPAR-regulated fatty acid oxidation genes such as CYP4A11, ACOX, UCP1 and ApoAI, and inhibition of LXRalpha-regulated lipogenesis genes, such as FAS, ABCA1, ABCG1, and HMGR. This effect results in the induction of a fasted-like state in primary rat hepatocytes in which fatty acid oxidation increases, while cholesterol and bile acid production decreases. Our findings explain the myriad effects of naringenin and support its continued clinical development. Of note, this is the first description of a non-toxic, naturally occurring LXRalpha inhibitor.

BACKGROUND: Environmental enrichment (EE) fosters attachment behavior through its effect on brain oxytocin levels in the hippocampus and other brain regions, which in turn modulate the hypothalamic-pituitary axis (HPA). Social isolation and other stressors negatively impact physical healing through their effect on the HPA. Therefore, we reasoned that: 1) provision of a rat EE (nest building with Nestlets) would improve wound healing in rats undergoing stress due to isolation rearing and 2) that oxytocin would have a similar beneficial effect on wound healing. METHODOLOGY/PRINCIPAL FINDINGS: In the first two experiments, we provided isolation reared rats with either EE or oxytocin and compared their wound healing to group reared rats and isolation reared rats that did not receive Nestlets or oxytocin. In the third experiment, we examined the effect of Nestlets on open field locomotion and immediate early gene (IEG) expression. We found that isolation reared rats treated with Nestlets a) healed significantly better than without Nestlets, 2) healed at a similar rate to rats treated with oxytocin, 3) had decreased hyperactivity in the open field test, and 4) had normalized IEG expression in brain hippocampus. CONCLUSIONS/SIGNIFICANCE: This study shows that when an EE strategy or oxytocin is given to isolation reared rats, the peripheral stress response, as measured by burn injury healing, is decreased. The findings indicate an association between the effect of nest making on wound healing and administration of the pro-bonding hormone oxytocin. Further elucidation of this animal model should lead to improved understanding of how EE strategies can ameliorate poor wound healing and other symptoms that result from isolation stress.

Modulation of the immune system may be a viable alternative in the treatment of fulminant hepatic failure (FHF) and can potentially eliminate the need for donor hepatocytes for cellular therapies. Multipotent bone marrow-derived mesenchymal stem cells (MSCs) have been shown to inhibit the function of various immune cells by undefined paracrine mediators in vitro. Yet, the therapeutic potential of MSC-derived molecules has not been tested in immunological conditions in vivo. Herein, we report that the administration of MSC-derived molecules in two clinically relevant forms-intravenous bolus of conditioned medium (MSC-CM) or extracorporeal perfusion with a bioreactor containing MSCs (MSC-EB)-can provide a significant survival benefit in rats undergoing FHF. We observed a cell mass-dependent reduction in mortality that was abolished at high cell numbers indicating a therapeutic window. Histopathological analysis of liver tissue after MSC-CM treatment showed dramatic reduction of panlobular leukocytic infiltrates, hepatocellular death and bile duct duplication. Furthermore, we demonstrate using computed tomography of adoptively transferred leukocytes that MSC-CM functionally diverts immune cells from the injured organ indicating that altered leukocyte migration by MSC-CM therapy may account for the absence of immune cells in liver tissue. Preliminary analysis of the MSC secretome using a protein array screen revealed a large fraction of chemotactic cytokines, or chemokines. When MSC-CM was fractionated based on heparin binding affinity, a known ligand for all chemokines, only the heparin-bound eluent reversed FHF indicating that the active components of MSC-CM reside in this fraction. These data provide the first experimental evidence of the medicinal use of MSC-derived molecules in the treatment of an inflammatory condition and support the role of chemokines and altered leukocyte migration as a novel therapeutic modality for FHF.

Abstract Chimeric Antigen Receptor (CAR) T-cell therapy is a highly effective treatment for B-cell malignancies but limited in use due to clinically significant hyperinflammatory toxicities. Understanding the pathophysiologic mechanisms which mediate these toxicities can help identify novel management strategies. Here we report a novel in vitro model of the macrophage-endothelial interface to study the effects of CAR T-cell-induced cytokine storm. Using this model, we demonstrate that macrophage-mediated inflammation is regulated by endothelial cell activity. Furthermore, endothelial inflammation occurs independently of macrophages following exposure to CAR T-cell products and the induced endothelial inflammation potentiates macrophage-mediated inflammatory signaling, leading to a hyperinflammatory environment. While corticosteroids, the current gold standard of care, attenuate the resulting macrophage inflammatory signaling, the endothelial activity remains refractory to this treatment strategy. Utilizing a network model, coupled to in vitro secretion profiling, we identified STAT3 programming as critical in regulating this endothelial behavior. Lastly, we demonstrate how targeting STAT3 activity can abrogate endothelial inflammation and attenuate this otherwise hyperinflammatory environment. Our results demonstrate that endothelial cells play a central role in the pathophysiology of CAR T-cell toxicities and targeting the mechanisms driving the endothelial response can guide future clinical management.

Despite decades of efforts, state-of-the-art synthetic burn dressings to treat partial-thickness burns are still far from ideal. Current dressings adhere to the wound and necessitate debridement. This work describes the first “supramolecular hybrid hydrogel (SHH)” burn dressing that is biocompatible, self-healable, and on-demand dissoluble for easy and trauma-free removal, prepared by a simple, fast, and scalable method. These SHHs leverage the interactions of a custom-designed cationic copolymer via host-guest chemistry with cucurbit[7]uril and electrostatic interactions with clay nanosheets coated with an anionic polymer to achieve enhanced mechanical properties and fast on-demand dissolution. The SHHs show high mechanical strength (>50 kPa), self-heal rapidly in ∼1 min, and dissolve quickly (4–6 min) using an amantadine hydrochloride (AH) solution that breaks the supramolecular interactions in the SHHs. Neither the SHHs nor the AH solution has any adverse effects on human dermal fibroblasts or epidermal keratinocytes in vitro. The SHHs also do not elicit any significant cytokine response in vitro. Furthermore, in vivo murine experiments show no immune or inflammatory cell infiltration in the subcutaneous tissue and no change in circulatory cytokines compared to sham controls. Thus, these SHHs present excellent burn dressing candidates to reduce the time of pain and time associated with dressing changes.

Objective: Osteoarthritis (OA) is a chronic joint disease, with limited treatment options, characterized by inflammation and matrix degradation, and resulting in severe pain or disability. Progressive inflammatory interaction among key cell types, including chondrocytes and macrophages, leads to a cascade of intra- and inter-cellular events which culminate in OA induction. In order to investigate these interactions, we developed a multi-cellular in vitro OA model, to characterize OA progression, and identify and evaluate potential OA therapeutics in response to mediators representing graded levels of inflammatory severity. Methods: We compared macrophages, chondrocytes and their co-culture responses to “low” Interleukin-1 (IL-1) or “high” IL-1/tumor necrosis factor (IL-1/TNF) levels of inflammation. We also investigated response changes following the administration of dexamethasone (DEX) or mesenchymal stromal cell (MSC) treatment via a combination of gene expression and secretory changes, reflecting not only inflammation, but also chondrocyte function. Results: Inflamed chondrocytes presented an osteoarthritic-like phenotype characterized by high gene expression of pro-inflammatory cytokines and chemokines, up-regulation of ECM degrading proteases, and down-regulation of chondrogenic genes. Our results indicate that while MSC treatment attenuates macrophage inflammation directly, it does not reduce chondrocyte inflammatory responses, unless macrophages are present as well. DEX however, can directly attenuate chondrocyte inflammation. Conclusions: Our results highlight the importance of considering multi-cellular interactions when studying complex systems such as the articular joint. In addition, our approach, using a panel of both inflammatory and chondrocyte functional genes, provides a more comprehensive approach to investigate disease biomarkers, and responses to treatment.

Background & Aims: Alterations in mitochondrial morphology and function and increased oxidative stresses in hepatocytes are well established in nonalcoholic fatty liver disease (NAFLD). Patients can undergo lifestyle changes, especially in earlier NAFLD stages, to reverse disease-induced phenotypes on a gross level. Yet, little is known about whether mitochondrial function and injuries recover upon reversal. Thus, we elucidated this question and interplays between the cytoskeletal network and mitochondria in the development and reversal of steatosis. Methods: We cultured primary human hepatocytes stably for 2 weeks and used free fatty acid supplementation to induce steatosis over 7 days and reversed steatosis by free fatty acid withdrawal over the next 7 days. We assessed cytoskeletal and mitochondrial morphologies using immunocytochemistry and confocal microscopy. We evaluated mitochondrial respiration and function via the Seahorse analyzer, in which we fully optimized reagent dosing specifically for human hepatocytes. Results: During early steatosis, intracellular lipid droplets displaced microtubules altering mitochondrial distribution, and disrupted the F-actin network, leading to loss of bile canaliculi in steatotic hepatocytes. Basal mitochondrial respiration, maximum respiratory capacity, and resistance to H2O2-induced cell death also increased as an adaptative response. Upon reversal of steatosis, F-actin and bile canaliculi were restored in hepatocytes. Nevertheless, we observed an increase in elongated mitochondrial branches accompanied by decreases in α-tubulin expression, mitochondrial proton leak, and susceptibility to H2O2-induced cell death. Conclusions: Despite the restoration of cytoskeletons morphologically upon reversal of steatosis, the mitochondria in hepatocytes were impaired owing to early adaptative respiratory increase. Hepatocytes thus were highly predisposed to H2O2-induced cell death. These results indicate the persistence of potential health risks for recovering NAFLD patients.

Background: Adenosine triphosphate (ATP) levels guide many aspects of the red blood cell (RBC) hypothermic storage lesions. As a result, efforts to improve the quality of hypothermic-stored red cell concentrates (RCCs) have largely centered around designing storage solutions to promote ATP retention. Considering reduced temperatures alone would diminish metabolism, and thereby enhance ATP retention, we evaluated: (a) whether the quality of stored blood is improved at −4°C relative to conventional 4°C storage, and (b) whether the addition of trehalose and PEG400 can enhance these improvements.Study Design and Methods: Ten CPD/SAGM leukoreduced RCCs were pooled, split, and resuspended in a next-generation storage solution (i.e., PAG3M) supplemented with 0–165 mM of trehalose or 0–165 mM of PEG400. In a separate subset of samples, mannitol was removed at equimolar concentrations to achieve a fixed osmolarity between the additive and non-additive groups. All samples were stored at both 4°C and −4°C under a layer of paraffin oil to prevent ice formation.Results: PEG400 reduced hemolysis and increased deformability in −4°C-stored samples when used at a concentration of 110 mM. Reduced temperatures did indeed enhance ATP retention; however, in the absence of an additive, the characteristic storage-dependent decline in deformability and increase in hemolysis was exacerbated. The addition of trehalose enhanced this decline in deformability and hemolysis at −4°C; although, this was marginally alleviated by the osmolarity-adjustments. In contrast, outcomes with PEG400 were worsened by these osmolarity adjustments, but at no concentration, in the absence of these adjustments, was damage greater than the control.Discussion: Supercooled temperatures can allow for improved ATP retention; however, this does not translate into improved storage success. Additional work is necessary to further elucidate the mechanism of injury that progresses at these temperatures such that storage solutions can be designed which allow RBCs to benefit from this diminished rate of metabolic deterioration. The present study suggests that PEG400 could be an ideal component in these solutions.

Electroporation involves applying electric field pulses to cells, leading to the alteration or destruction of cell membranes. Irreversible electroporation (IRE) creates permanent defects in cell membranes and induces cell death. By directly targeting IRE to tumors, percutaneous nonthermal ablation is possible. The history of IRE, evolution of concepts, theory, biological applications, and clinical data regarding its safety and efficacy are discussed.