Biomedical subjects

Martin L Yarmush

Publications and source records attributed to Martin L Yarmush.

At least 19 recordsLinked to original sources

PubMed2005

Identification of neutrophil gelatinase-associated lipocalin (NGAL) as a discriminatory marker of the hepatocyte-secreted protein response to IL-1beta: a proteomic analysis.

The liver is the major source of proteins used throughout the body for various functions. Upon injury or infection, an acute phase response (APR) is initiated in the liver that is primarily mediated by inflammatory cytokines such as interleukin-1beta (IL-1beta) and interleukin-6. Among others, the APR is characterized by an altered protein synthetic profile. We used two-dimensional gel electrophoresis to study the dynamics of changes in protein synthesis in hepatocytes exposed to these inflammatory cytokines. Protein profiles were quantified using image analysis and further analyzed using multivariate statistical methods. Our results indicate that IL-1beta and IL-6 each induces secreted protein responses with distinct dynamics and dose-dependence. Parallel stimulation by IL-1beta and IL-6 results in a protein pattern indistinguishable from the IL-1beta pattern, indicating a dominant effect of IL-1beta over IL-6 at the doses tested. Multidimensional scaling (MDS) of correlation distances between protein secretion levels revealed two protein pairs that are robustly co-secreted across the various cytokine stimulation conditions, suggesting shared regulatory pathways. Finally, we also used multivariate alternating conditional expectation (MACE) to identify transformation functions that discriminated the cytokine-stimulated and untreated hepatocyte-secreted protein profiles. Our analysis indicates that the expression of neutrophil gelatinase-associated lipocalin (NGAL) was sufficient to discriminate between IL-1beta and IL-6 stimulation. The combination of proteomics and multivariate analysis is expected to provide new information on the cellular regulatory networks involved in generating specific cellular responses.

Acute-Phase Proteins↗

PubMed2005

Microfabricated grooved substrates as platforms for bioartificial liver reactors.

An extracorporeal bioartificial liver device has the potential to provide temporary hepatic support for patients with liver failure. Our goal was to optimize the flow environment for the cultured hepatocytes in a flat-plate bioreactor, specifically focusing on oxygen delivery using high medium flow rates while reducing the detrimental effects of the resulting shear stresses. We used photolithographic techniques to fabricate microgrooves onto the underlying glass substrate. The microgrooves, perpendicular to the axial flow direction, protected the hepatocytes from the shear stress induced by the flowing medium. Using finite element analysis, we found that the velocity gradient change near the cell surface (i.e., bottom of the grooves) was smaller than that near the top surface of the flow channel, indicating that the grooves would provide protection to the attached cells from the mechanical effects of the flowing medium. We also determined that the shear stress at the cell surface could be reduced by as much as 30 times (channel height of 100 microm) in the grooved-substrate (0.5 dyn/cm(2)) bioreactor compared to the flat-substrate (15 dyn/cm(2)) bioreactor for a medium flow rate of 4.0 mL/min. Albumin and urea synthesis rates of hepatocytes cocultured with 3T3-J2 fibroblasts remained stable over 5 days of perfusion in the grooved-substrate bioreactor, whereas in the flat-substrate bioreactor they decreased over the same time period. These studies indicate that under "high" flow conditions the microgrooved-substrate in the bioreactor can decrease the detrimental effects of shear stress on the hepatocytes while providing adequate oxygenation, thereby resulting in stable liver-specific function.

3T3 Cells↗

PubMed2005

Immunodepletion of albumin for two-dimensional gel detection of new mouse acute-phase protein and other plasma proteins.

Immunodepletion of albumin to improve the 2-D gel resolution of human plasma proteins has recently been described. With the importance of mouse models in many studies in which serum or plasma is often analyzed, we have adopted this approach to immunoprecipitate mouse albumin and evaluated its effectiveness for 2-D separation of mouse plasma proteins. Purified polyclonal antibodies against mouse albumin were effective depleting intact albumin as well as its numerous fragments from mouse plasma samples. Removal of albumin resulted in better resolution of mouse plasma proteins. Three proteins, alpha2-macroglobulin, coagulation factor XII, and hemopexin, that were previously either undetectable or poorly resolved, were identified from albumin-depleted 2-D gels by peptide mass fingerprinting. Albumin depletion also led to partial loss of several other proteins such as clusterin and gelsolin. This loss can be attributed to the interaction with albumin itself because the specificity of the antibody was demonstrated by Western blot. When applying this method to the 2-D separation of plasma from inflamed mouse induced by cutaneous burn injury with superimposed Pseudomonas aeruginosa infection, the upregulation of inter alpha-trypsin inhibitor heavy chain 4 (ITIH4) and hemopexin was unambiguously detected along with other mouse acute-phase proteins (APP), including haptoglobin and serum amyloid A. Based on the significant increase of ITIH4, we propose that this protein is a new member of mouse APP that are upregulated during the inflammatory response.

Acute-Phase Proteins↗

PubMed2005

Optimization of reporter cells for expression profiling in a microfluidic device.

The emergence of green fluorescence protein (GFP) technologies has enabled non-invasive monitoring of cell function and gene expression. GFP-based expression studies are typically performed in traditional single-dish or multi-well formats to monitor a small number of genes or conditions that do not lend well to scaling, high-throughput analysis, or single-cell measurements. We have recently developed a microfluidic device, the Living Cell Array (LCA), for monitoring GFP-based gene expression in a high-throughput manner. Here, we report the optimization of GFP reporter cell characteristics in this microfluidic device for gene expression profiling. A reporter cell line for the transcription factor NF-kappa B was generated and used as the model cell line. Reporter cells were seeded in the LCA and NF-kappa B activated by addition of the cytokine TNF-alpha . Our studies show that the fluorescence kinetics from the reporter cell line in response to both single and repeated TNF-alpha stimulation in the LCA is similar to that observed in standard tissue culture. In addition, our data also indicate that multiple expression waves can be reliably monitored from a small population of reporter cells. Using reporter cell line subcloning and cell cycle synchronization, we demonstrate that the kinetics and magnitude of induced fluorescence in the reporter cell lines can be further improved to maximize the fluorescence readout from reporter cell lines, thereby improving their applicability to live cell expression profiling. Our studies establish some of the important criteria to be considered when using reporter cell lines for dynamic expression profiling in microfluidic devices.

Cell Culture Techniques↗

PubMed2005

Effects of dehydroepiandrosterone administration on rat hepatic metabolism following thermal injury.

BACKGROUND: Severe burns cause dramatic alterations in liver and whole-body metabolism. Recently, there has been interest in using dehydroepiandrosterone (DHEA) as a treatment for trauma patients, and enhanced survival and immune function have been reported using DHEA in animal trauma models. The specific effects of DHEA on hepatic metabolism following burn injury have not been explored. MATERIALS AND METHODS: Male rats received either (1) a burn covering approximately 20% of the total body surface area or a sham burn or (2) burn injury followed by two intraperitoneal injections of DHEA or vehicle. After 4 days, the livers were isolated and perfused in vitro, and 28 metabolite fluxes were measured. Metabolic flux analysis was used to obtain the intracellular metabolic flux distribution and provide an overview of the metabolic state of the livers in each experimental group. RESULTS: Burn injury decreased the uptake of lactate and the production of beta-hydroxybutyrate and increased the deamination of glutamine to glutamate and asparagine to aspartate. DHEA, compared to vehicle treatment, decreased pentose phosphate pathway (PPP) fluxes and the uptake of several amino acids in burned rats. Furthermore, DHEA treatment restored liver metabolism in burned rats to a state that was very similar to that of the sham control group. CONCLUSIONS: DHEA administration appears to normalize hepatocellular metabolism in burned rats but also decreases the PPP flux, which may impair the liver's ability to recycle endogenous antioxidants. DHEA treatment combined with exogenous antioxidants should receive further consideration in the management of burn and trauma patients.

Adjuvants, Immunologic↗

PubMed2005

Evolution of intrahepatic carbon, nitrogen, and energy metabolism in a D-galactosamine-induced rat liver failure model.

A clearer picture of the hepatic metabolic pathways affected by fulminant hepatic failure (FHF) would help develop nutritional support and nonsurgical therapies for FHF. We characterized the evolution of hepatic metabolism in a rat model of FHF using an isolated perfused liver system together with a mass-balance model of intermediary metabolism. Principal component analysis (PCA) was used to identify potential new sensitive markers for FHF. To induce FHF, rats were given two D-galactosamine injections under fasting conditions. Controls were fasted only. Livers were harvested 1, 4, 8, and 12 h later and perfused with Eagle minimal essential medium supplemented with amino acids and bovine serum albumin, and equilibrated with 95% O2/5% CO2. At the 1 h time point, lactate release increased concomitant with a decrease in gluconeogenesis, TCA cycle and mitochondrial electron transport fluxes. At 4 h, amino acid metabolism and urea cycle fluxes were significantly depressed. By 8 h, gluconeogenesis had switched to glycolysis. By 12 h, amino acid metabolism was broadly inhibited, and there was a net release of many amino acids. Mass-balance analysis shows that the main source of ATP production in the FHF liver gradually changed from mitochondrial oxidative phosphorylation to glycolysis. PCA suggests that a linear combination of glucose, lactate, and glutamine concentrations in arterial plasma is a sensitive marker for FHF. We conclude that D-galactosamine causes early mitochondrial dysfunction while glycolytic ATP synthesis remains functional. Markers that are indirectly linked to these pathways may be used to evaluate the progression of FHF.

Animals↗

PubMed2004

Quantitative effects of thermal injury and insulin on the metabolism of the skeletal muscle using the perfused rat hindquarter preparation.

Injury from a severe burn or trauma can propel the body into a hypermetabolic state that can lead to the significant erosion of lean muscle mass. Investigations describing this process have been somewhat limited due to the lack of adequate experimental models. Here we report the use of a perfused rat hindquarter preparation to study the consequences of a moderate burn injury (approximately 20% total body surface area), with or without the addition of exogenous insulin (12.5 mU/mL), on the fluxes of major metabolites across the isolated skeletal muscle. The metabolic flux data was further analyzed using metabolic flux analysis (MFA), which allows for the estimation of the impact of these conditions on the intracellular muscle metabolism. Results indicate that this model is able to capture the increased rate of proteolysis, glutamine formation, and the negative nitrogen balance associated with the burn-induced hypermetabolic state. The inclusion of exogenous insulin resulted in significant changes in several fluxes, including an increase in the metabolism of glucose and the flux through the pentose phosphate pathway, as well as a reduction in the metabolism of glutamine, alanine, and leucine. However, insulin administration did not affect the nitrogen balance or the rate of proteolysis in the muscle, as has been suggested using other techniques. The use of the perfused hindquarter model coupled with MFA is a physiologically relevant and experimentally flexible platform for the exploration of skeletal muscle metabolism under catabolic conditions, and it will be useful in quantifying the specific metabolic consequences of other therapeutic advances.

Animals↗

PubMed2004

Dynamic gene expression profiling using a microfabricated living cell array.

We describe the development of a microfluidic platform for continuous monitoring of gene expression in live cells. This optically transparent microfluidic device integrates high-throughput molecular stimulation with nondestructive monitoring of expression events in individual living cells, hence, a living cell array (LCA). Several concentrations of a soluble molecular stimulus are generated in an upstream microfluidic network and used to stimulate downstream reporter cells, each containing a green fluorescence reporter plasmid for a gene of interest. Cellular fluorescence is continuously monitored and quantified to infer the expression dynamics of the gene being studied. We demonstrate this approach by profiling the activation of the transcription factor NF-kappaB in HeLa S3 cells in response to varying doses of the inflammatory cytokine TNF-alpha. The LCA platform offers a unique opportunity to simultaneously control dynamic inputs and measure dynamic outputs from adherent mammalian cells in a high-throughput fashion. This approach to profiling expression dynamics, in conjunction with complementary techniques such as DNA microarrays, will help provide a more complete picture of the dynamic cellular response to diverse soluble stimuli.

Gene Expression Profiling↗

PubMed2004

Designing a hepatocellular microenvironment with protein microarraying and poly(ethylene glycol) photolithography.

In this study, robotic protein printing was employed as a method for designing a cellular microenvironment. Protein printing proved to be an effective strategy for creating micropatterned co-cultures of primary rat hepatocytes and 3T3 fibroblasts. Collagen spots (ca. 170 microm in diameter) were printed onto amino-silane- and glutaraldehyde-modified glass slides. Groups of 15-20 hepatocytes attached to collagen regions in a highly selective manner forming cell clusters corresponding in size to the printed collagen domains. Fibroblasts, seeded onto the same surface, adhered and spread around arrays of hepatocyte islands creating a heterotypic environment. The co-cultured hepatocytes produced and maintained high levels of liver-specific biomarkers, albumin and urea, over the course of 2 weeks. In addition, protein printing was combined with poly(ethylene glycol) photolithography to define intercellular contacts within the clusters of hepatocytes residing on individual collagen islands. Glass slides, treated with 3-acryloxypropyl trichlorosilane and imprinted with 170 m diameter collagen spots, were micropatterned with a high-density array of 30 microm x 30 microm poly(ethylene glycol) (PEG) wells. As a result, discrete groups of ca. 9 PEG microwells became functionalized with the cell-adhesive ligand. When exposed to micropatterned surfaces, hepatocytes interacted exclusively with collagen-modified regions, attaching and becoming confined at a single-cell level within the hydrogel wells. Micropatterning strategies proposed here will lead to greater insights into hepatocellular behavior and will benefit the fields of hepatic tissue engineering and liver biology.

3T3 Cells↗

PubMed2004

Growth factors and nonparenchymal cell conditioned media induce mitogenic responses in stable long-term adult rat hepatocyte cultures.

Most prior studies have characterized hepatocyte proliferative responses in culture systems that do not express a stable differentiated phenotype. We investigated the DNA synthetic response of long-term stable hepatocyte cultures to growth factor stimulation as well as conditioning with nonparenchymal cells (NPCs). Primary rat hepatocytes were cultured on a single layer of collagen (h/C) or Matrigel (h/M), or in a collagen sandwich (C/h/C) or collagen-Matrigel sandwich (M/h/C). Hepatocytes were cultured for 7 days to allow phenotypic stabilization before growth factor addition, except for h/C cultures, which are unstable, where growth factors were added 1 day after seeding. Culture medium was supplemented with a mixture of hepatocyte, epidermal, and vascular endothelial growth factors and interleukin-6, either directly or after conditioning with NPCs for 24 h. Growth factors alone induced hepatocyte DNA synthesis, as measured via [3H]thymidine uptake, in the h/C, C/h/C, and M/h/C configurations. h/M exhibited very low levels of DNA synthesis. In the C/h/C and M/h/C configurations, the greatest stimulation was obtained using NPC-conditioned growth factors. This response was sustained for several days and without decreasing albumin or urea synthesis. These results suggest that hepatocyte mitogens and NPC-derived factors can stimulate DNA synthesis in stable and differentiated hepatocyte cultures.

Albumins↗

PubMed2004

Identification of optimal classification functions for biological sample and state discrimination from metabolic profiling data.

MOTIVATIONS: Classification of biological samples for diagnostic purposes is a difficult task because of the many decisions involved on the number, type and functional manipulations of the input variables. This study presents a generally applicable strategy for systematic formulation of optimal diagnostic indexes. To this end, we develop a novel set of computational tools by integrating regression optimization, stepwise variable selection and cross-validation algorithms. RESULTS: The proposed discrimination methodology was applied to plasma and tissue (liver) metabolic profiling data describing the time progression of liver dysfunction in a rat model of acute hepatic failure generated by d-galactosamine (GalN) injection. From the plasma data, our methodology identified seven (out of a total of 23) metabolites, and the corresponding transform functions, as the best inputs to the optimal diagnostic index. This index showed better time resolution and increased noise robustness compared with an existing metabolic index, Fischer's BCAA/AAA molar ratio, as well as indexes generated using other commonly used discriminant analysis tools. Comparison of plasma and liver indexes found two consensus metabolites, lactate and glucose, which implicate glycolysis and/or gluconeogenesis in mediating the metabolic effects of GalN.

Algorithms↗

PubMed2004

A mouse serum two-dimensional gel map: application to profiling burn injury and infection.

With the importance of mouse as a model to study human diseases and the human and rat plasma/serum two-dimensional (2-D) maps being extensively annotated, this study was aimed at constructing a detailed mouse serum 2-D map. Serum proteins from two different inbred strains of mice (BALB/cJ and C57BL/6J) and mice subjected to two different inflammatory stimuli (20% burn injury and lipopolysaccharide (LPS) injection) were separated on overlapping gels covering pH 3-8 and stained with SYPRO Ruby dye. The tryptic peptides from the resolved spots were analyzed by mass spectrometry, leading to the identification of 38 different gene products. With the exception of major urinary proteins found in abundance in male C57BL/6J mice, little strain difference of the mouse serum 2-D was observed. Many proteins detected in the mouse serum 2-D map were not reported in human or rat serum 2-D maps including epidermal growth factor receptor. Three major murine acute-phase proteins (APPs), haptoglobin, serum amyloid A, and serum amyloid P, were highly induced by both inflammatory stimuli. Image analysis shows that the variations of APPs between these two inflammatory models were not uniform although LPS (100 microg/animal) in general was more effective than 20% burn injury in inducing APPs. Serum amyloid A, much more sensitive to endotoxin than burn injury, may represent a sensitive marker to differentiate these two different inflammatory states.

Animals↗

PubMed2004

Technique for expanding the donor liver pool: heat shock preconditioning in a rat fatty liver model.

Fatty liver is a common predisposing risk factor for postoperative liver failure and accounts for most discarded livers during triage of donors. We investigated the effect of heat shock preconditioning (HPc) on recipient survival in a rat fatty liver transplantation model. Fatty liver donor rats were exposed to brief whole-body hyperthermia (10 minutes at 42.5 degrees C) and allowed to recover. HPc induced heat shock proteins (HSPs) (HSP72, HSP90, and heme oxygenase [HO]-1) in donor livers, with levels peaking 12 to 48 hours after HPc. Subsequently, donor livers were harvested 24 hours after HPc, placed in cold storage for 10 hours, and transplanted into normal rats. At 3 hours posttransplantation, HPc reduced serum liver enzymes in the recipients and almost completely suppressed the release of tumor necrosis factor (TNF)-alpha and interleukin (IL)-10. Histologic evaluation 3 and 24 hours after transplantation showed that HPc significantly reduced hepatic inflammation and hepatocellular necrosis without affecting the steatotic appearance of hepatocytes. One week after transplantation, control non-heat-shocked and heat-shocked fatty liver recipients exhibited survival rates of less than 10% and more than 80%, respectively. The evaluation of the survival of recipients receiving fatty livers at different times after HPc showed that the protective effect of HPc was significant when donor livers were transplanted 3 to 48 hours after HPc, with the maximum effect seen 6 to 48 hours after HPc. In conclusion, HPc is a promising avenue to salvage rejected donor fatty livers and enhance the survival rate of fatty liver recipients. We estimate that this technique could increase the annual donor pool by 600 livers.

Animals↗

PubMed2004

Hepatic tissue engineering for adjunct and temporary liver support: critical technologies.

The severe donor liver shortage, high cost, and complexity of orthotopic liver transplantation have prompted the search for alternative treatment strategies for end-stage liver disease, which would require less donor material, be cheaper, and less invasive. Hepatic tissue engineering encompasses several approaches to develop adjunct internal liver support methods, such as hepatocyte transplantation and implantable hepatocyte-based devices, as well as temporary extracorporeal liver support techniques, such as bioartificial liver assist devices. Many tissue engineered liver support systems have passed the "proof of principle" test in preclinical and clinical studies; however, they have not yet been found sufficiently reliably effective for routine clinical use. In this review we describe, from an engineering perspective, the progress and remaining challenges that must be resolved in order to develop the next generation of implantable and extracorporeal devices for adjunct or temporary liver assist.

Biomedical Technology↗

PubMed2004

Dispensable role for interferon-gamma in the burn-induced acute phase response: a proteomic analysis.

We examined the role of the pleiotropic cytokine interferon-gamma (IFN-gamma) in initiating the burn injury-induced acute phase response (APR). Two-dimensional (2-D) electrophoresis was used to obtain serum protein profiles from wild-type (WT) and IFN-gamma knockout mice following sham-burn or 20% burn injury. Serum 2-D images from both groups of burn-injured mice were characterized by the upregulation of a similar panel of protein spots. These included the three major murine acute phase proteins haptoglobin, serum amyloid A, and serum amyloid P, that were identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry. Furthermore, the changes in the levels of these protein spots were very similar between these two groups of mice, as determined by image analysis. Other features of burn-induced APR such as a decrease in total serum protein concentration, an elevated circulation level of the cytokine interleukin-6 (IL-6), and activation of the IL-6 signal transduction protein STAT3 were also evaluated and found to be similar between wild-type and IFN-gamma knockout mice. These results suggest a dispensable role of IFN-gamma in the induction of the hepatic APR in mice following burn injury.

Acute-Phase Reaction↗

PubMed2004

Charged polymers modulate retrovirus transduction via membrane charge neutralization and virus aggregation.

The specific mechanisms of charged polymer modulation of retrovirus transduction were analyzed by characterizing their effects on virus transport and adsorption. From a standard colloidal perspective two mechanisms, charge shielding and virus aggregation, can potentially account for the experimentally observed changes in adsorption behavior and biophysical parameters due to charged polymers. Experimental testing revealed that both mechanisms could be at work depending on the characteristics of the cationic polymer. All cationic polymers enhanced adsorption and transduction via charge shielding; however, only polymers greater than 15 kDa in size were capable of enhancing these processes via the virus aggregation mechanism, explaining the higher efficiency enhancement of the high molecular weight molecules. The role of anionic polymers was also characterized and they were found to inhibit transduction via sequestration of cationic polymers, thereby preventing charge shielding and virus aggregation. Taken together, these findings suggest the basis for a revised physical model of virus transport that incorporates electrostatic interactions through both virus-cell repulsive and attractive interactions, as well as the aggregation state of the virus.

Animals↗

PubMed2004

Quantitative dynamics of in vivo bone marrow neutrophil production and egress in response to injury and infection.

Production rates of blood cells from the bone marrow (BM) can be determined from pool size and residence time in the circulation only during steady state. We describe a method to evaluate changes in BM neutrophil production following severe injury. Male CD-1 mice underwent nonlethal cutaneous burn injury, a lethal burn injury with Pseudomonas aeruginosa infection, or sham treatment, and received bromodeoxyuridine (BrdU) to label proliferative cells. Rates of BM neutrophil production and release into the circulation were determined using a mathematical model that integrates BM neutrophil pool size and fraction of BrdU labeled cells as a function of time. Absolute rates could not be quantified without BrdU data for the neutrophil progenitor pool; however, relative rates could be determined. BM neutrophil production and release significantly increased after injury. After nonlethal burn, release transiently exceeded production, causing a temporary decrease in BM neutrophil stores followed by reestablishment of a steady-state BM neutrophil pool similar to sham controls. After lethal burn infection, release always exceeded production, causing complete depletion of BM neutrophils and suppression of BM neutrophil production. This method is generally applicable to estimating production rates of nonproliferating, terminally differentiated cells, arising from a stem cell pool in vivo.

Animals↗

PubMed2003

Profiling of dynamic changes in hypermetabolic livers.

The liver plays an important role in the overall negative nitrogen balance leading to muscle wasting commonly observed in patients following many conditions, including severe injury, cancer, and diabetes. In order to study changes in liver metabolism during the establishment of such catabolic states, we used a rat skin burn injury model that induces hypermetabolism and muscle wasting. At various times during the first week following the injury, livers were isolated and perfused in a recirculating system under well-defined conditions. We applied a steady-state metabolic flux analysis model of liver metabolism and then used k-means clustering to objectively group together reaction flux time profiles. We identified six distinct groups of reactions that were differentially responsive: (1) pentose phosphate pathway (PPP); (2) amino acid oxidation reactions leading to the formation of tricarboxylic acid (TCA) cycle intermediates; (3) gluconeogenesis; (4) TCA-cycle and mitochondrial oxidation; (5) lipolysis, beta-oxidation, and ketone body formation; and (6) urea-cycle. Burn injury sequentially upregulated the urea-cycle, the PPP, and the TCA-cycle, in order, while beta-oxidation and gluconeogenesis remained unchanged. The upregulation of the PPP was transient, whereas the rise in urea- and TCA-cycle fluxes was sustained. An ATP balance predicted an increased production of ATP and energy expenditure starting on day 3 post-burn, which correlated with the induction of the oxidative phosphorylation uncoupler uncoupling protein-2. We conclude that metabolic profiling using flux analysis and clustering analysis is a useful methodology to characterize the differential activation of metabolic pathways in perfused organs and to identify specific key pathways that are sensitive to a stimulus or insult without making a priori assumptions.

Adaptation, Physiological↗