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We studied the electric response of fractures with laboratory experiments and numerical simulations for a full-bore formation microimaging tool. The laboratory setup was designed and built to perform controlled experiments with accurate measurements of all principal properties involved for electric borehole imaging. These properties are formation resistivity, mud resistivity, fracture aperture, pad position, and button current. The experiments were conducted on two types of limestone for fracture apertures ranging from 0.1 to 0.9 mm and mud/formation resistivity contrasts varying from 1/100 to 1/10,000. A numerical model was used to reproduce the laboratory configuration and to validate the results. The model proved to be an effective tool to optimize the experimental setup, and it was also used to study the effect of standoff (up to 5 mm) on the measured integrated additional current. Linear relationships between the fracture aperture and measured integrated current were found to be valid for the laboratory experiment and the corresponding numerical simulation. The measured integrated current could therefore be used to determine the fracture aperture if the other parameters are known. Two coefficients in the relationship were found to differ from those previously found using numerical simulations for the actual borehole situation. These differences are attributed to tool- and scale-dependent factors.

Understanding the regulatory networks which control specific macrophage phenotypes is essential in identifying novel targets to correct macrophage mediated clinical disorders, often accompanied by inflammatory events. Since mesenchymal stromal cells (MSCs) have been shown to play key roles in regulating immune functions predominantly via a large number of secreted products, we used a fractional factorial approach to streamline experimental evaluation of MSC mediated inflammatory macrophage regulation. Our macrophage reprogramming metrics, human bone marrow MSC attenuation of macrophage pro-inflammatory M1 TNFα secretion and simultaneous enhanced expression of the M2 macrophage marker, CD206, were used as analysis endpoints. Objective evaluation of a panel of MSC secreted mediators indicated that PGE2 alone was sufficient in facilitating macrophage reprogramming, while IL4 only provided partial reprogramming. Inhibiting stromal cell PGE2 secretion with Indomethacin, reversed the macrophage reprogramming effect. PGE2 reprogramming was mediated through the EP4 receptor and indirectly through the CREB signaling pathway as GSK3 specific inhibitors induced M1 macrophages to express CD206. This reprogramming pathway functioned independently from the M1 suppression pathway, as neither CREB nor GSK3 inhibition reversed PGE2 TNF-α secretion attenuation. In conclusion, fractional factorial experimental design identified stromal derived PGE2 as the factor most important in facilitating macrophage reprogramming, albeit via two unique pathways.

Macrosteatotic livers exhibit elevated intrahepatic triglyceride (TG) levels in the form of large lipid droplets (LDs), reduced adenosine triphosphate (ATP) levels, and elevated reactive oxygen species (ROS) levels, and this contributes to their elevated sensitivity to ischemia/reperfusion injury during transplantation. Reducing macrosteatosis in living donors through dieting has been shown to improve transplant outcomes. Accomplishing the same feat for deceased donor grafts would require ex vivo exposure to potent defatting agents. Here we used a rat hepatocyte culture system exhibiting a macrosteatotic LD morphology, elevated TG levels, and an elevated sensitivity to hypoxia/reoxygenation (H/R) to test for such agents and ameliorate H/R sensitivity. Macrosteatotic hepatocyte preconditioning for 48 hours with a defatting cocktail that was previously developed to promote TG catabolism reduced the number of macrosteatotic LDs and intracellular TG levels by 82% and 27%, respectively, but it did not ameliorate sensitivity to H/R. Supplementation of this cocktail with l-carnitine, together with hyperoxic exposure, yielded a similar reduction in the number of macrosteatotic LDs and a 57% reduction in intrahepatic TG storage, likely by increasing the supply of acetyl coenzyme A to mitochondria, as indicated by a 70% increase in ketone body secretion. Furthermore, this treatment reduced ROS levels by 32%, increased ATP levels by 27% (to levels near those of lean controls), and completely abolished H/R sensitivity as indicated by approximately 85% viability after H/R and the reduction of cytosolic lactate dehydrogenase release to levels seen in lean controls. Cultures maintained for 48 hours after H/R were approximately 83% viable and exhibited superior urea secretion and bile canalicular transport in comparison with untreated macrosteatotic cultures. In conclusion, these findings show that the elevated sensitivity of macrosteatotic hepatocytes to H/R can be overcome by defatting agents, and they suggest a possible route for the recovery of discarded macrosteatotic grafts.

There is a critical shortage of transplantable livers in both the US and the world. One method to increase the donor pool is to develop methodology to recondition extended criteria donor grafts, a large portion of which are moderate to severe macrosteatotic livers. Transplantation of these livers often leads to primary nonfunction caused by an increased susceptibility to the effects of ischemia reperfusion injury that result from the harvesting, transportation, and transplantation of the liver. Our lab has developed a novel cocktail of defatting reagents that can, over a period of two days, render hepatocytes lean and with good cell viability and function. Despite this accomplishment, in order to be feasibly performed in a clinical setting, the defatting process must be completed in a matter of hours. The current project focuses on understanding the differences between defatting in a static versus flow environment and then using this information to develop the ideal parameters for defatting whole organs. Our hypothesis in the current work is that using flow and the appropriate defatting agents, steatotic hepatocytes can be defatted in a clinically relevant time of hours, without decreasing cell viability or function. In order to test this hypothesis, a perfusion reactor was constructed which holds microscope slides that can be seeded with fatty hepatocytes. Perfusion of defatting media was performed at different flow rates over a six hour period. Results showed that within a six hour period, significant defatting (as compared to static cultures) was observed at both 2.0 and 4.0 ml/min as indicated by oil red O staining. Current experiments are focused on further evaluation of hepatocyte viability and function (e.g. albumin, urea, and cytochrome P450 function). These types of parametric experiments will form the basis for future perfusions with whole organs from obese rats.

Large-droplet macrovesicular steatosis (ld-MaS) in more than 30% of liver graft hepatocytes is a major risk factor for liver transplantation. An accurate assessment of the ld-MaS percentage is crucial for determining liver graft transplantability, which is currently based on pathologists' evaluations of hematoxylin and eosin (HE)-stained liver histology specimens, with the predominant criteria being the relative size of the lipid droplets (LDs) and their propensity to displace a hepatocyte's nucleus to the cell periphery. Automated image analysis systems aimed at objectively and reproducibly quantifying ld-MaS do not accurately differentiate large LDs from small-droplet macrovesicular steatosis and do not take into account LD-mediated nuclear displacement; this leads to a poor correlation with pathologists' assessments. Here we present an improved image analysis method that incorporates nuclear displacement as a key image feature for segmenting and classifying ld-MaS from HE-stained liver histology slides. 52,000 LDs in 54 digital images from 9 patients were analyzed, and the performance of the proposed method was compared against the performance of current image analysis methods and the ld-MaS percentage evaluations of 2 trained pathologists from different centers. We show that combining nuclear displacement and LD size information significantly improves the separation between large and small macrovesicular LDs (specificity = 93.7%, sensitivity = 99.3%) and the correlation with pathologists' ld-MaS percentage assessments (linear regression coefficient of determination = 0.97). This performance vastly exceeds that of other automated image analyzers, which typically underestimate or overestimate pathologists' ld-MaS scores. This work demonstrates the potential of automated ld-MaS analysis in monitoring the steatotic state of livers. The image analysis principles demonstrated here may help to standardize ld-MaS scores among centers and ultimately help in the process of determining liver graft transplantability.

The Formation MicroImager (mark of Schlumberger) is an electric imaging tool that produces electrical scans of the borehole walls. These measurements provide useful information on the fracture aperture of naturally fractured reservoirs. In this paper, we present a laboratory set-up that was realized to perform controlled experiments on fractured samples using a Formation MicroImager pad. A three-dimensional numerical model was used to develop the laboratory set-up. Numerical simulations were run to investigate the relationship between the fracture aperture and its electrical response for different properties (mud resistivity, formation resistivity and tool standoff). Preliminary results showed the capability of the set-up to be used to perform controlled experiments for a wide range of fracture properties and investigate their influence on the determination of the fracture aperture.

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 6days in fatty acid-supplemented medium to induce steatosis, and then switched for 2days 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 3days and macrosteatosis in 6days, 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 2days. 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.

Macrovesicular steatosis in greater than 30% of hepatocytes is a significant risk factor for primary graft nonfunction due to increased sensitivity to ischemia reperfusion (I/R) injury. The growing prevalence of hepatic steatosis due to the obesity epidemic, in conjunction with an aging population, may negatively impact the availability of suitable deceased liver donors. Some have suggested that metabolic interventions could decrease the fat content of liver grafts prior to transplantation. This concept has been successfully tested through nutritional supplementation in a few living donors. Utilization of deceased donor livers, however, requires defatting of explanted organs. Animal studies suggest that this can be accomplished by ex vivo warm perfusion in a time scale of a few hours. We estimate that this approach could significantly boost the size of the donor pool by increasing the utilization of steatotic livers. Here we review current knowledge on the mechanisms whereby excessive lipid storage and macrosteatosis exacerbate hepatic I/R injury, and possible approaches to address this problem, including ex vivo perfusion methods as well as metabolically induced defatting. We also discuss the challenges ahead that need to be addressed for clinical implementation.

Advanced drug delivery nanosystems (aDDnSs) combining liposomal and dendritic materials have only recently appeared in the research field of drug delivery. The nature and localization of the interactions between the components of such systems are not yet fully described. In this study, liposomes are combined with hyperbranched polyesters for the development of new aDDnSs. The polymer - lipid interactions along with their dependence on the polyesters pseudogeneration number and the liposomal lipid composition have been examined. The results indicate that the interaction between the materials takes place in the headgroup region, where H-bonds between the polymers terminal hydroxyls and the phospholipids phosphate moiety are formed. Due to the polymers' compact imperfect structure, which varies with pseudogeneration number, no linear trends are observed with increasing pseudogeneration number. Moreover, it is shown that high percentages of cholesterol in the lipid bilayer affect the penetration of the polymers in the headgroup region. © 2011 American Chemical Society.