Biomedical subjects

R G Tompkins

Publications and source records attributed to R G Tompkins.

At least 109 records · Page 6Linked to original sources

PubMed1994

An extracorporeal microscopy perfusion chamber for on-line studies of environmental effects on cultured hepatocytes.

The development of bioartificial-hybrid organ support systems is hampered by the lack of knowledge on the effects of different (in vivo) environments on cells during extracorporeal perfusion. In the present study, a perfusion chamber was designed for continuous monitoring of cultured cells during perfusion with media, as well as during plasma perfusion in an extracorporeal circuit. Chamber characterization showed satisfactory thermal and perfusion profiles and no major pH fluctuations. Further testing was performed with hepatocytes that were cultured in between two collagen layers, a configuration which was previously shown to preserve hepatocyte morphology and function for over six weeks of culture. Perfusion of the hepatocytes with culture media did not adversely affect cell morphology and function, provided the perfusion time was < or = 48 hours. Perfusion of the cultures during connection of the chamber to an extracorporeal circuit involving normal rats for six hours resulted in reversible cytoplasmic changes, unaltered cell shapes indices, and a 40 percent increase in albumin secretion rate during the first post-perfusion day, followed by a return to stable control levels. We expect that this chamber will be a valuable tool for on-line studies of cells under (extracorporeal) perfusion conditions and could be used for a large variety of studies on regeneration, reperfusion damage, and detoxification.

Albumins↗

PubMed1994

A general model for analysis of the tricarboxylic acid cycle with use of [13C]glutamate isotopomer measurements.

A generalized steady-state model was developed for determining tricarboxylic acid cycle fractional fluxes from 13C nuclear magnetic resonance (NMR) data. The model relates the measured mole fractions of [13C]glutamate isotopomers to the fractional fluxes and predicted mole fractions of isotopomers of oxaloacetate (OAA) and acetyl-CoA. This model includes cycling between OAA and fumarate. Fractional fluxes are determined by fitting the model equations to NMR parameters by use of nonlinear least squares. Although only fractional fluxes can be determined from 13C-NMR data, when they are combined with mass spectroscopic measurements, absolute values can be derived. A specific metabolic system represented by published 13C-NMR data from extracts of hearts perfused with [13C]acetate, [13C]pyruvate (PYR), and [13C]acetate plus [13C]PYR was used to test the model. The intensities of predicted 13C-NMR splitting patterns were compared with observed values, and there was excellent agreement between observed and predicted signal intensities. With this model, important physiological parameters, including the OAA-derived fraction of inflow to PYR, PYR-derived fraction of inflow to acetyl-CoA, citrate-derived fraction of inflow to OAA, and PYR-derived fraction of inflow to OAA, can be determined.

Animals↗

PubMed1994

Management of burn wounds with prompt excision and immediate closure.

The past 30 years have been witness to significant improvements in the overall care and prognosis of those suffering burn trauma. At the heart of this success is an aggressive approach to burn wounds. This approach, which is detailed in this review, involves early operative removal of devitalized tissue and biological coverage of resultant wounds, with particular attention to wounds in specialized areas.

Administration, Topical↗

PubMed1994

Prophylactic antibiotics and their role in the prevention of surgical wound infection.

Prophylactic antibiotics are indicated in all clean-contaminated and many clean procedures, especially in view of the low complication rates seen with cephalosporin prophylaxis. They are best administered immediately before operation in a manner that insures a tissue level at the time of incision and they should target pathogens commonly associated with the specific operation undertaken. Therapeutic tissue concentrations should be maintained throughout the period of potential bacterial contamination but not beyond 24 hours after surgery. Linton has been proved corrected in predicting the contribution of preventive (prophylactic) antibiotics to surgery. They are a useful tool in the surgeon's effort to minimize the occurrence of postoperative infection. However, these agents must be used with, and cannot replace, meticulous and aseptic surgical technique. Current research providing information concerning the basic cellular and molecular bases of host defense against bacterial primary lodgment and invasion provide strong evidence that the surgical patient of the future will benefit by an extended ability to normalize natural host defenses rendering those few bacteria that gain access to the wound incapable of producing infection. At that time preventive (prophylactic) antibiotics will become less important in the control of postoperative infection.

Anti-Bacterial Agents↗

PubMed1994

Thermal injury induces very early production of interleukin-1 alpha in the rat by mechanisms other than endotoxemia.

BACKGROUND: Cytokines are putative mediators of thermal injury-induced systemic changes. We studied the effects of thermal injury on cytokine activation in vivo with a sensitive radioimmunoassay specific for rat interleukin-1 alpha (IL-1 alpha). METHODS: We characterized the organ distribution and expression kinetics of IL-1 alpha in rats submitted to either 20% total body surface area cutaneous burn, muscle burn, or endotoxic shock. Rats were killed at various time points, and liver, lung, spleen, ileum, thymus, kidney, skin, and plasma were harvested. Tissues were homogenized, and the supernates were assayed for rat IL-1 alpha. The assay detection limit was 1.5 ng/gm wet tissue (WT). RESULTS: Thermal injury induced marked elevations of IL-1 alpha levels in the liver and lung, and maximal levels were reached at 2.5 hours when compared with controls. In the liver mean IL-1 alpha levels in cutaneous burn injury were 16.5 +/- 6.2 ng/gm WT, whereas in sham injury they were 1.7 +/- 0.1 ng/gm WT, p < or = 0.05; in the lung IL-1 alpha levels with cutaneous burn injury were 10.3 +/- 1.3 ng/gm WT, whereas sham injury levels were 1.9 +/- 0.8 ng/gm WT, p < or = 0.002). Levels in all other organs and plasma were below detection limits. Muscle burn injury had similar elevated levels of IL-1 alpha in the liver at 1 hour, indistinguishable from cutaneous burn. In contrast, endotoxin challenge resulted in dramatic elevation of IL-1 alpha levels in all organs tested except for the kidney, whereas the skin maintained its usual large amounts of IL-1 alpha. CONCLUSIONS: These data indicate that thermal or mechanical injury induce very early and organ-specific association of IL-1 alpha in vivo by mechanisms other than endotoxemia.

Animals↗

PubMed1994

Antisense DNA delivery in vivo: liver targeting by receptor-mediated uptake.

UNLABELLED: Antisense oligodeoxynucleotides coupled to asialoglycoprotein carrier molecules were evaluated in terms of their ability to accumulate preferentially in the liver and thus potentially serve as an important method to regulate liver gene expression. METHODS: Native and asialo-human alpha-1 acid glycoproteins were derivatized with low molecular weight poly(L)lysine and complexed with an antisense DNA (67 mer) complementary to the 5' end of rat serum albumin mRNA. The asialoglycoprotein antisense complex (conjugate) was characterized with respect to size, stability, and anti-sense loading, and the biodistribution of the conjugate was determined for normal rats at 5 min and 1, 6, and 24 hr after intravenous injection. In vivo stability of the anti-sense asialoglycoprotein complex was also evaluated using double-labeled (32P-antisense and 3H-glycoprotein) preparations. RESULTS: The results of the conjugate characterization studies demonstrated that at least 30% of the anti-sense DNA dissociated from the carrier after 7 min under chromatographic conditions. When the conjugate was incubated with PBS, MEM or MEM plus 10% FBS for 1 hr at 37 degrees C, about 85% of the antisense DNA was dissociated from the carrier. The results of the biodistribution studies showed that the accumulation of the asialo-glycoprotein anti-sense complex in the liver was rapid and greatly exceeded the accumulation of the sialo-glycoprotein antisense analog or antisense alone. CONCLUSION: These findings have significant implications for the targeted delivery of therapeutic antisense molecules to the liver.

Animals↗

PubMed1993

Proline-mediated enhancement of hepatocyte function in a collagen gel sandwich culture configuration.

Isolated rat primary hepatocytes were cultured between two layers of gelled collagen in a sandwich configuration that reinstates the cellular polarity necessary for long-term function in vitro. Maintenance of hepatocyte function, as measured by the secretion of albumin, was shown to be dependent on both the sandwich gel configuration and the continued presence of L-proline in the culture media. Cis-hydroxyproline, an analog of proline known to prevent the proper folding of triple helical collagen molecules, inhibited the response of sandwiched hepatocytes to proline in a dose-dependent and reversible manner. The addition of cis-hydroxyproline to cultures established for 7 days also resulted in the inhibition of hepatocyte function. These data support the hypothesis that continued collagen synthesis by hepatocytes is critical for hepatocyte function in the sandwich gel configuration.

Animals↗

PubMed1993

Effect of collagen gel configuration on the cytoskeleton in cultured rat hepatocytes.

The cytoskeleton is important in the maintenance of cellular morphology and differentiated function in a number of cell types, including hepatocytes. In this study, adult rat hepatocytes sandwiched between two layers of collagen gel were compared to cells cultured on a single collagen gel for differences in the organization and expression of the cytoskeletal proteins actin and tubulin. Hepatocytes cultured between two layers of hydrated rat tail tendon collagen (sandwich gel) morphologically resembled cells in intact liver for several weeks. Actin filaments (F-actin) in these hepatocytes were concentrated under the plasma membrane in regions of cell-cell contact. In contrast, hepatocytes cultured on a single collagen gel were flattened and motile and had F-actin containing stress fibers. This was accompanied by a severalfold increase in actin mRNA. Microtubules formed an interwoven network in hepatocytes cultured in a sandwich gel, but in single gel cultures they formed long parallel arrays extending out to the cell periphery. Tubulin mRNA was severalfold greater in hepatocytes cultured on a single gel. Fibronectin and laminin staining were greater in single gel cultures, and these proteins were concentrated in fibrils radiating from the cell periphery. Overlaying a second collagen gel onto hepatocytes that had been cultured on a single gel (double gel rescue) reversed cell spreading and reduced stress fibers. Double gel rescue also resulted in a decrease in actin and tubulin mRNA to levels present in sandwich gel cultures and freshly isolated hepatocytes. These results show that the configuration of the external matrix has a dynamic effect on cytoskeletal proteins in cultured rat hepatocytes.

Actins↗

PubMed1993

Intestinal production of interleukin-1 alpha during endotoxemia in the mouse.

Interleukin-1 (IL-1) may be involved in gut permeability to macromolecules and gut glutamine metabolism during endotoxemia. We developed a sensitive radioimmunoassay specific for mouse IL-1 alpha (detection limit of 100 pg/ml, or 5 pM) and measured intestinal levels of IL-1 alpha in response to endotoxin. CD-1 mice (N = 190) were randomized to intraperitoneal (ip) or intravenous (i.v.) lipopolysaccharide (LPS) infusion (15 micrograms/g or 1.5 micrograms/g Escherichia coli 0111:B4 LPS) or saline. Mice were sacrificed at Time 0, 30 min, 1 hr, 2.5 hr, 4 hr, 6 hr, 12 hr, and 24 hr (3 mice/group/time point). Small bowel (SB) and large bowel (LB) were harvested and compared to liver. Duodenum, upper jejunum, midjejunum, terminal ileum, cecum, ascending colon, and sigmoid were analyzed in separate experiments. Tissues were frozen, weighed, and homogenized, the homogenates were centrifuged, and the supernates were assayed for immunoreactive IL-1 alpha. IL-1 alpha was expressed as pg/g wt +/- SEM (lowest detectable amount = 1000 pg/g wet tissue (WT)). SB but not LB from normal controls had constitutively elevated levels of IL-1 alpha (6177 +/- 1640 pg/g WT). LPS ip or i.v. produced lethargy, diarrhea, and a dramatic elevation of IL-1 alpha levels in both SB and LB. In SB, IL-1 alpha was elevated compared to baseline at 1 hr (19201 +/- 626 pg/g WT) and reached a fivefold maximal increase at 2.5 hr (31775 +/- 503 pg/g WT) following 15 micrograms/g ip.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Gut macromolecular permeability in pancreatitis correlates with severity of disease in rats.

BACKGROUND: Increased intestinal macromolecular permeability could allow absorption of substances from the bowel into the systemic circulation and contribute to multiple organ system failure. METHODS: Mild, intermediate, and severe grades of pancreatitis were induced in rats using intravenous caerulein and intraductal glycodeoxycholic acid. [14C]polyethylene glycol (molecular weight, 3350 daltons; 1.1 microCi/142 mg) was instilled into the distal duodenum. At 24 hours, the animals were killed, ascitic fluid was collected for trypsinogen activation peptide measurement, and pancreatic specimens were collected and scored for based on the degree of necrosis, inflammation, and hemorrhage. RESULTS: Gut permeability to polyethylene glycol 3350 (PEG 3350) was increased in animals with early experimental pancreatitis (5.4% +/- 1.2%, n = 20) when compared with control animals (1.8% +/- 0.2%; n = 6) (P = 0.0005). Furthermore, intestinal macromolecular permeability to PEG 3350 correlated with severity of disease as predicted by the method of induction of pancreatitis (P = 0.0003), the histological findings (P = 0.0002), and total ascitic trypsinogen activation peptides content (P = 0.029). CONCLUSIONS: Increased gut permeability in experimental pancreatitis can be correlated with pancreatitis severity.

Animals↗

PubMed1993

Immobilized IL-2 preserves the viability of an IL-2 dependent cell line.

The mouse T-cell line CTLL-2 is known to be dependent on interleukin-2 (IL-2) for both growth and viability. These cells possess high affinity IL-2 receptors and have been shown to internalize IL-2 after binding. To determine if internalization of IL-2 is required for the mediation of its signal, IL-2 was covalently coupled to an insoluble matrix via glutaraldehyde cross-linking and CTLL-2 cells were incubated with the immobilized lymphokine matrix. This covalent cross-linking prevents the free lateral diffusion and internalization of the bound IL-2 receptors (IL-2R) while still permitting specific binding between the cells and the immobilized ligands. Although only very limited proliferation was observed during the incubation as assessed by 3H-thymidine incorporation, the viability of the CTLL-2 cells on the immobilized IL-2 matrix was preserved. Cells incubated on the immobilized IL-2 surface could proliferate in response to exogenous soluble IL-2 that was added to the cultures after 36 hours whereas control cultures incubated with an immobilized BSA matrix had died. This indicates that immobilized IL-2 can mediate some of the activity of soluble IL-2 and that internalization of the IL-2 receptor may not be required for at least part of the IL-2 mediated effect.

Animals↗

PubMed1993

Absence of change in hepatic lactate metabolism after burn injury.

In response to severe injury, extraordinary quantities of lactate that are released from the peripheral tissues serve as substrate for hepatic gluconeogenesis. It is possible that as a result of burn injury, reaction kinetics involving lactate could be directly changed within the liver. The metabolic fate of [U-14C]lactate was examined in vitro in fresh liver slices after a 20 per cent total body surface area injury. Glucose and CO2 were produced in vitro by the liver tissues after the injury and no differences were seen in the metabolism of these substrates between the injured and control animals. These findings suggest that the intrinsic enzymatic processes within the liver are not directly altered by injury itself or by any of the associated inflammatory mediators which appear early after burns.

Animals↗

PubMed1993

Acute adrenal insufficiency in the burn intensive care unit.

Acute adrenal insufficiency may underlie a confusing, stormy, intensive care unit course after a burn. The aetiology is most likely to be adrenal haemorrhage despite the absence of a coagulopathy. This report describes two patients who were diagnosed antemortem and successfully treated with replacement therapy. The aetiology, presentation, diagnosis and treatment of acute adrenal insufficiency in the intensive care unit is reviewed.

Acute Disease↗

PubMed1993

Nucleation and growth of ice crystals inside cultured hepatocytes during freezing in the presence of dimethyl sulfoxide.

A three-part, coupled model of cell dehydration, nucleation, and crystal growth was used to study intracellular ice formation (IIF) in cultured hepatocytes frozen in the presence of dimethyl sulfoxide (DMSO). Heterogeneous nucleation temperatures were predicted as a function of DMSO concentration and were in good agreement with experimental data. Simulated freezing protocols correctly predicted and explained experimentally observed effects of cooling rate, warming rate, and storage temperature on hepatocyte function. For cells cooled to -40 degrees C, no IIF occurred for cooling rates less than 10 degrees C/min. IIF did occur at faster cooling rates, and the predicted volume of intracellular ice increased with increasing cooling rate. Cells cooled at 5 degrees C/min to -80 degrees C were shown to undergo nucleation at -46.8 degrees C, with the consequence that storage temperatures above this value resulted in high viability independent of warming rate, whereas colder storage temperatures resulted in cell injury for slow warming rates. Cell damage correlated positively with predicted intracellular ice volume, and an upper limit for the critical ice content was estimated to be 3.7% of the isotonic water content. The power of the model was limited by difficulties in estimating the cytosol viscosity and membrane permeability as functions of DMSO concentration at low temperatures.

Animals↗

PubMed1993

Nonequilibrium freezing of one-cell mouse embryos. Membrane integrity and developmental potential.

A thermodynamic model was used to evaluate and optimize a rapid three-step nonequilibrium freezing protocol for one-cell mouse embryos in the absence of cryoprotectants (CPAs) that avoided lethal intracellular ice formation (IIF). Biophysical parameters of one-cell mouse embryos were determined at subzero temperatures using cryomicroscopic investigations (i.e., the water permeability of the plasma membrane, its temperature dependence, and the parameters for heterogeneous IIF). The parameters were then incorporated into the thermodynamic model, which predicted the likelihood of IIF. Model predictions showed that IIF could be prevented at a cooling rate of 120 degrees C/min when a 5-min holding period was inserted at -10 degrees C to assure cellular dehydration. This predicted freezing protocol, which avoided IIF in the absence of CPAs, was two orders of magnitude faster than conventional embryo cryopreservation cooling rates of between 0.5 and 1 degree C/min. At slow cooling rates, embryos predominantly follow the equilibrium phase diagram and do not undergo IIF, but mechanisms other than IIF (e.g., high electrolyte concentrations, mechanical effects, and others) cause cellular damage. We tested the predictions of our thermodynamic model using a programmable freezer and confirmed the theoretical predictions. The membrane integrity of one-cell mouse embryos, as assessed by fluorescein diacetate retention, was approximately 80% after freezing down to -45 degrees C by the rapid nonequilibrium protocol derived from our model. The fact that embryos could be rapidly frozen in the absence of CPAs without damage to the plasma membrane as assessed by fluorescein diacetate retention is a new and exciting finding. Further refinements of this protocol is necessary to retain the developmental competence of the embryos.

Animals↗

PubMed1993

Technetium-99m-labeled hydrazino nicotinamide derivatized chemotactic peptide analogs for imaging focal sites of bacterial infection.

We synthesized and evaluated four hydrazino nicotinamide (HYNIC) derivatized chemotactic peptide analogs: For-NleLFK-HYNIC (HP1), For-MLFK-HYNIC (HP2), For-MLFNH(CH2)6NH-HYNIC (HP3), and For-MLF-(D)-K-HYNIC (HP4), for in vitro bioactivity and receptor binding. The peptides were radiolabeled with 99mTc via a glucoheptonate co-ligand and their biodistribution determined in rats (n = 6/time point) at 5, 30, 60 and 120 min after injection. Localization of the peptides at sites of deep thigh Escherichia coli infection was determined by radioactivity measurements on excised tissues in rats (n = 6/time point) and rabbits as well as scintillation camera imaging in rabbits (n = 6). All peptides maintained biological activity (EC50s for O2 production by human PMNs: 12-500 nM) and the ability to bind to the oligopeptide chemoattractant receptor on human PMNs (EC50s for binding: 0.12-40 nM). After incubation with 99mTc-glucoheptonate, radiolabeled peptides were isolated by HPLC at specific activities of > 10,000 mCi/microM. Technetium-99m-labeled peptides retained receptor binding with EC50s < 10 nM. Blood clearance of all four peptides was rapid. Biodistributions of the individual peptides were similar, with low levels of accumulation in most normal tissues. In rats, all of the peptides concentrated at the infection sites (T/B ratio: 2.5-3:1) within 1 hr of injection. In rabbits, outstanding images of the infection sites were obtained, with T/B ratios of > 20:1 at 15 hr after injection. This study demonstrates that 99mTc-labeled chemotactic peptide analogs are effective agents for the external imaging of focal sites of infection.

Animals↗

PubMed1993

In vivo bioactivity and biodistribution of chemotactic peptide analogs in nonhuman primates.

The dose dependence of the effect of chemotactic peptide on peripheral leukocyte levels was measured in normal Rhesus monkeys. A 99mTc-labeled hydrazino nicotinamide (HYNIC) derivatized chemotactic peptide analog was used to study biodistribution and inflammation imaging in Rhesus monkeys. In normal animals the studies demonstrated that chemotactic peptide induced a clear dose-dependent reduction in peripheral leukocyte levels. The decrease in leukocyte number occurred almost immediately after injection and rapidly returned to baseline. Significant effects on differential WBC count, blood pressure, pulse rate or respiration rate were not detected. The lowest dose of peptide tested (10 ng/kg) had minimal effect on leukocyte level. The HYNIC derivatized peptide was prepared in excellent yield and purity, had biological activity similar to the native peptide and was readily labeled at specific activity of > 20,000 mCi/mumole. When approximately 0.5 mCi (< 2.0 ng/kg) of radiolabeled peptide was injected in monkeys with focal sites of mild sterile inflammation, a pattern of biodistribution similar to radiolabeled WBCs was observed and reductions in leukocyte levels were not detected. At 3 hr after injection, the site of inflammation was readily apparent with a target-to-background ratio of approximately 3:1. These studies demonstrate that radiolabeled chemotactic peptide analogs are effective agents for imaging sites of inflammation in monkeys. By radiolabeling at high specific activity, the effect of these reagents on peripheral leukocyte levels can be avoided.

Animals↗

PubMed1993

Technetium-99m-labeled chemotactic peptides: comparison with indium-111-labeled white blood cells for localizing acute bacterial infection in the rabbit.

The biodistribution and infection imaging properties of 99mTc-labeled formyl-methionyl-leucyl-phenylalanyl-lysyl-hydrazinonicotinamide (99mTc-HP) were compared with 111In-labeled leukocytes (111In-WBCs) in rabbits with E. coli infections. Groups of six animals were co-injected with 1 mCi of 99mTc-HP plus 0.05 mCi of 111In-WBCs and serial scintigrams were acquired from 3 to 6 hr and 18 hr postinjection. After acquiring the final images, the animals were killed and biodistribution was determined. At all imaging times, the distributions of 99mTc-HP and 111In-WBCs were similar and the sites of infection were well visualized with both radiopharmaceuticals. The target (infected muscle) to background (contralateral normal muscle) ratios (T/B) were: 3.38 +/- 0.46, 3.80 +/- 0.37 and 10.87 +/- 1.44 for 99mTc-HP and 1.71 +/- 0.04, 1.81 +/- 0.26 and 3.79 +/- 0.83, for 111In-WBCs at 3, 6 and 18 hr postinjection, respectively. The average ratio of T/B ratios (99mTc-HP-to-111In-WBCs) was 2.99 +/- 1.88, with no value less than unity. T/B ratios calculated from direct tissue sampling were significantly higher for 99mTc-HP than for 111In-WBCs (33.6:1 versus 8.1:1, p < 0.01). These differences were primarily due to increased absolute accumulation of 99mTc-HP (0.102%ID/g versus 0.024%ID/g, p < 0.01) in infected muscle rather than a difference in accumulation in normal skeletal muscle. These results indicate that 99mTc-HP yields target-to-background ratios greater than or equal to those achievable with 111In-WBCs probably as a result of an increase in absolute accumulation at the site of infection.

Acute Disease↗