Multisorbent plasma perfusion in fulminant hepatic failure: effects of duration and frequency of treatment in rats with grade III hepatic coma.

Using the model of galactosamine-induced fulminant hepatic failure in the rat, the effects of multisorbent plasma perfusion over Asahi uncoated spherical charcoal, Plasorba (BR-350) resin, and an endotoxin removing adsorbent (polymyxin B-sepharose) were determined in Grade III hepatic coma animals by studying survival as influenced by timing, duration, and frequency of treatment. The effects of treatment on liver cell proliferation and endotoxin removal also were examined. The results demonstrate that duration and frequency of treatment are major contributing factors in the successful application of nonbiological membrane-based multisorbent liver support systems. Examination of the regenerative activity in the liver indicates an enhanced proliferative response following multisorbent plasma perfusion compared with untreated fulminant hepatic failure (FHF) paired controls. Utilizing an endotoxin removal adsorbent alone, a marked reduction in systemic levels of endotoxin in FHF was demonstrated compared with nonperfused FHF paired controls. Despite current emphasis on bioartificial liver support systems, plasma purification by multisorbent systems offers a simple method for the removal of circulating toxic metabolites in general together with specific toxin removal.

Oxygen transfer in a convection-enhanced hollow fiber bioartificial liver.

A mathematical model was developed to predict oxygen transport in a hollow fiber bioartificial liver device. The model parameters were taken from the HepatAssist 2000 device, a plasma perfused hollow fiber cartridge with primary hepatocytes seeded in the extracapillary space. Cellular oxygen uptake was based on Michaelis-Menten kinetics. Oxygen transport due to the convective flow of plasma into the extracapillary space was considered. The effect of modulating several important parameters was investigated, namely, the Michaelis-Menten constant V(m) (the maximum oxygen consumption per unit volume of the cell mass), the oxygen partial pressure, the flow rate of the plasma at device inlet, and the permeability of the cell mass contained in the extracapillary space. A computer implementation of the model was used to assess whether a given number of cells could be maintained within such a device. The results suggest that a substantial proportion of the hepatocytes are exposed to hypoxic conditions under which metabolism may be impaired.

Oxygen transfer in a diffusion-limited hollow fiber bioartificial liver.

A mathematical model was developed to predict oxygen transport in a hollow fiber bioartificial liver device. Model parameters were taken from the Hepatix ELAD configuration; a blood perfused hollow fiber cartridge with hepatocytes seeded in the extracapillary space. Cellular oxygen uptake is based on Michaelis-Menten kinetics, and nonlinear oxygen transport in the blood is considered. The effect of modulating three important parameters is investigated, namely, the Michaelis-Menten constants Vm (volumetric oxygen consumption of the hepatocytes) and Km (half-saturation constant), and hollow fiber oxygen permeability. A computer implementation of the model is used to assess whether a given cell mass could be maintained within such a device. The results suggest that liver cell lines possessing low rates of oxygen consumption could be maintained if membranes of sufficiently high oxygen permeability are used. For primary hepatocytes, which have much higher oxygen demands, radial transport of oxygen is rate limiting, and the axial-flow hollow fiber cartridge is thus an inappropriate design for use as a bioartificial liver with primary hepatocytes.

In vitro investigation of the blood response to medical grade PVC and the effect of heparin on the blood response.

This paper reports the results of an investigation into the blood response of polymers in vitro, using non-anticoagulated and heparinised blood and plasma. The materials studied were regenerated cellulose, (Cuprophan), an acrylonitrile-allyl sulphonate copolymer (AN69S), and medical grade polyvinyl chloride plasticised with di-2-ethyl-hexyl-phthalate (PVC/DEHP). Blood-material or plasma-material contact was achieved using a parallel plate flow cell, and C3a generation and FXII-like activity measured. The results of the study with non-anticoagulated human blood show that PVC/DEHP is a high complement activator. C3a concentration in the blood was higher after contact with PVC/DEHP than after contact with regenerated cellulose. The introduction of heparin in the blood induced complex alterations in the blood response. C3a generation could be elevated, decreased, or remain the same, depending on the material. The FXII-like activity on the surface of the PVC/DEHP after contact with plasma was also higher than the other two polymers. The introduction of heparin could increase or decrease FXII-like activity, depending on material. The patterns of response obtained with non-anticoagulated blood in vitro for AN69S and Cuprophan bore a strong resemblance with patterns of response obtained in the clinic, whereas those obtained with heparinised blood in vitro did not.

Characterization of the Saccharomyces cerevisiae ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) involved in sterol biosynthesis.

All but two genes involved in the ergosterol biosynthetic pathway in Saccharomyces cerevisiae have been cloned, and their corresponding mutants have been described. The remaining genes encode the C-3 sterol dehydrogenase (C-4 decarboxylase) and the 3-keto sterol reductase and in concert with the C-4 sterol methyloxidase (ERG25) catalyze the sequential removal of the two methyl groups at the sterol C-4 position. The protein sequence of the Nocardia sp NAD(P)-dependent cholesterol dehydrogenase responsible for the conversion of cholesterol to its 3-keto derivative shows 30% similarity to a 329-aa Saccharomyces ORF, YGL001c, suggesting a possible role of YGL001c in sterol decarboxylation. The disruption of the YGL001c ORF was made in a diploid strain, and the segregants were plated onto sterol supplemented media under anaerobic growth conditions. Segregants containing the YGL001c disruption were not viable after transfer to fresh, sterol-supplemented media. However, one segregant was able to grow, and genetic analysis indicated that it contained a hem3 mutation. The YGL001c (ERG26) disruption also was viable in a hem 1Delta strain grown in the presence of ergosterol. Introduction of the erg26 mutation into an erg1 (squalene epoxidase) strain also was viable in ergosterol-supplemented media. We demonstrated that erg26 mutants grown on various sterol and heme-supplemented media accumulate nonesterified carboxylic acid sterols such as 4beta, 14alpha-dimethyl-4alpha-carboxy-cholesta-8,24-dien-3be ta-ol and 4beta-methyl-4alpha-carboxy-cholesta-8,24-dien-3beta-o l, the predicted substrates for the C-3 sterol dehydrogenase. Accumulation of these sterol molecules in a heme-competent erg26 strain results in an accumulation of toxic-oxygenated sterol intermediates that prevent growth, even in the presence of exogenously added sterol.

Cytotoxicity of bile in human Hep G2 cells and in primary cultures of rat hepatocytes.

There has been increasing interest in the development of a hepatocyte bioreactor for the treatment of acute hepatic failure; however, little is known about the effect of hepatocyte byproducts on the viability of the cells in the bioreactor environment. We investigated the effects of increasing concentrations of bile on the growth and viability of the human hepatoma cell line Hep G2 and on the cytochrome P-450 content and dependent mixed function oxidase (MFO) activities, reduced glutathione (GSH) content, and glutathione S-transferase (GST) activity of primary cultures of rat hepatocytes. Our purpose was to determine whether or not it would be necessary to pretreat the plasma from patients with acute liver failure to remove elevated bile concentrations which might be toxic to the hepatocytes in an artificial liver device. Bile was found to inhibit Hep G2 cell growth at concentrations as low as 0.1% and to decrease viability at concentrations above 0.5%. The cytochrome P-450 and GSH contents and the activities of the MFO system and of GST were decreased in the primary cultures of hepatocytes following 24 h treatment with concentrations of bile at and above 0.5%. The MFO activities associated with different cytochrome P-450 isoenzymes decreased to different extents in the presence of bile with the O-dealkylation of pentoxyresorufin being more labile than that of ethoxyresorufin. Our data indicate that elevated bile concentrations are cytotoxic to liver cells, and it may be necessary to pretreat patient plasma to decrease its bile content to protect the cells during the clinical operation of a hepatocyte bioreactor device.

The effects of serum from patients with acute liver failure on the growth and metabolism of Hep G2 cells.

In many bioartificial liver systems currently being designed and evaluated for use in fulminant hepatic failure, direct contact is required between the patient's blood and the liver cells in the device. The efficacy of such devices will be influenced by the interaction of fulminant hepatic failure (FHF) patient serum with the cells. We have found that FHF serum inhibits the growth rate and the synthesis of DNA, RNA, and protein; disturbs glutathione homeostasis; and induces morphological changes in cultured human Hep G2 cells. These interactions should influence the design of bioartificial liver devices based on proliferating cell lines and indicate the requirement to pretreat FHF patient plasma to reduce the toxin load.

Complement activation by cellulose: investigation of the effects of time, area, flow rate, shear rate and temperature on C3a generation in vitro, using a parallel plate flow cell.

The development and utilization of a parallel plate flow system to study the blood response to flat sheet biomaterials, is described. Unlike most other parallel plate flow systems, which have been used to study cellular interactions with biomaterials, the controlled flow test cell described below employs the test materials on both sides of the channel through which the blood flows. The flow cell is used to conduct an investigation into the in vitro generation of C3a by a regenerated cellulose membrane, Cuprophan. The effects of experimental variables such as temperature, blood flow rate, contact area and wall shear rate on C3a generation by Cuprophan were studied. The results show that C3a generation by Cuprophan is lower at 12 degrees C than at 22 degrees C, which is in turn lower than C3a generation at 37 degrees C. Furthermore, a decrease in contact area, and increase in wall shear rate and blood flow rate, can produce a decrease in C3a concentration.

In vitro blood compatibility evaluation of hollow fibre membrane using a controlled flow system: a comparative study.

A procedure has been established for the in vitro assessment of hollow fibre haemodialysis membranes. A 30 ml syringe containing 20 ml of fresh non-anticoagulated blood was mounted onto a non-pulsatile syringe pump and blood was perfused through minimodules constructed from 80 fibres retrieved from Cuprophan (Baxter ST15), cellulose acetate (M57-12, JMS Co Ltd, Hiroshima, Japan), and AN69HF (Filtral 20, Hospal, France) dialysers. Samples were collected before perfusion, 3, 6, 9 and 12 minutes. The modules were clamped vertically to minimise the effect of red cell pooling and the dialysate compartment was filled with 0.9% saline to minimise ultrafiltration. After sample processing, complement C3a, thrombin-antithrombin III complexes, prothrombin F1 + 2, and factor XII-like activity were evaluated. The results indicated that the system could discriminate between the membranes evaluated and therefore was a relevant procedure for the assessment of hollow fibre haemodialysis membranes.

Water vapour transmission rates in burns and chronic leg ulcers: influence of wound dressings and comparison with in vitro evaluation.

One of the main functions of wound dressings is to control water vapour transmission rate (WVTR) from wounded skin. In this paper, the influence of hydrocolloid, knitted viscose and gauze dressings was evaluated through in vivo measurement of WVTR in burns and chronic leg ulcers utilizing an evaporimeter. The results suggest that the evaporative water vapour loss from exposed skin wounds depends mainly on the wound depth, and that chronic leg ulcers have the same level of the WVTR as full thickness burns. Compared with the knitted viscose and gauze dressings, hydrocolloid dressing has a greater effect on reducing evaporative water loss, with WVTR being 20-30% of that of exposed wounds under the conditions used in this study. This result is in agreement with that obtained in an in vitro evaluation.

In vitro uptake and elimination of isoflurane by different membrane oxygenators.

OBJECTIVE: This study was designed to investigate the effect of membrane oxygenator design and composition on the uptake and elimination of isoflurane. DESIGN: Prospective, in vitro laboratory study. SETTING: Bioengineering laboratory. PARTICIPANTS: Three types of membrane oxygenator were tested: the SM-35 (polydimethylsiloxane in sheet form), the CML (polypropylene in sheet form), and the SAFE II (polypropylene in hollow-fiber form). The oxygenators were incorporated into a standard cardiopulmonary bypass circuit. INTERVENTIONS: Isoflurane was added to the oxygenator input gas and measured in exhaust gas and in (bovine) blood leaving the oxygenator at 1, 2, 3, 5, 7, 10, 15, and 20 minutes. The isoflurane vaporizer was then turned off, and samples were obtained at the same time intervals. The experiment was performed at 28 degrees C and 37 degrees C. MEASUREMENTS AND MAIN RESULTS: Uptake and elimination of isoflurane were slower via the SM-35 compared with the CML and the SAFE II (p < 0.01). CONCLUSIONS: If isoflurane is administered during cardiopulmonary bypass, knowledge of the influence of oxygenator membrane composition on its pharmacokinetics is essential if patient awareness and unexpected cardiovascular depression are to be avoided.

Techniques for measurement of oxygen consumption rates of hepatocytes during attachment and post-attachment.

Three techniques for measuring oxygen consumption rate (OCR) of cultured cells relevant to the development of bioartificial liver devices are reported. In an oxystat apparatus, HepG2 cells immobilised on Cytodex 3 microcarriers at a concentration of 10(6) cells ml-1 had a mean OCR of 0.7 nmol s-1/10(6) cells. The OCR decreased with increasing cell density, a characteristic previously reported for other cell lines. Rat hepatocytes immobilised on single collagen layers in a flow cell and challenged with ammonia had a mean OCR of 0.59 nmol s-1/10(6) cells. A novel two-compartment oxystat system was used to determine the OCR of rat hepatocytes during the attachment phase. OCR declined from 1.0 nmol s-1/10(6) immediately after seeding to 0.7 nmol s-1/10(6) cells at nine hours. The low OCR for HepG2 reflects loss of certain oxygen dependent metabolic pathways. The OCR measured for rat hepatocytes during and post-attachment are significantly higher than those reported elsewhere and have major implications for the development of bioartificial liver devices.

In vitro contact phase activation with haemodialysis membranes: role of pharmaceutical agents.

Contact phase activation was investigated in vitro using flat sheet type of haemodialysis membranes, Cuprophan (Akzo, Faser, Germany) and AN69S (Hospal, France), and a negatively charged polyamide Ultipor NR 14225 membrane as a control. The investigation focussed on the determination of factor XII-like activity (FXIIA) as an indicator of contact phase activation in the supernatant phase and at the membrane surface after plasma-membrane contact using an incubation test cell. The findings were compared with the observations from a plasma-free system utilizing purified unactivated factor XII. The plasma FXIIA bound to the membrane surface was significantly different between the membranes, while the supernatant phase FXIIA exhibited no significant differences. In contrast, the plasma-free system exhibited significant differences in the supernatant FXIIA and membrane-bound FXIIA for all the materials used and the magnitude of the activity was significantly greater for negatively charged materials. This finding demonstrated the strong influence of the interaction of other plasma constituents on the membrane surface and as such the binding and subsequent activation of factor XII may be altered possibly due to competitive binding and steric hindrance. On the addition of anticoagulants such as heparin, low-molecular-weight heparin, citrate and hirudin, no significant differences were observed in plasma supernatant phase FXIIA. However, each anticoagulant appears to have a distinct influence on the magnitude of plasma membrane-bound FXIIA. On the addition of aprotinin (a kallikrein inhibitor), no significant differences were observed in the plasma supernatant FXIIA. In contrast, aprotinin appears to significantly reduce membrane-bound FXIIA on Cuprophan and polyamide NR, but significantly increase the magnitude of the membrane-bound FXIIA on AN69S.

Cholesterol biosynthesis from lanosterol: regulation and purification of rat hepatic sterol 14-reductase.

We have previously characterized the membrane-bound sterol 14-reductase (14-reductase) that catalyzes anaerobically NADPH-dependent reduction of the 14-double bond of delta 8,14-diene or delta 7,14-diene sterols that are sterol intermediates in cholesterol biosynthesis in mammals (Paik et al. (1984) J. Biol. Chem. 259, 13413-13423). To elucidate the regulatory mechanism as well as molecular characteristics of the 14-reductase, we extended our investigation on the consequences of alteration of the enzymic activity under various physiological conditions. The enzymic activity of rat hepatic sterol 14-reductase was induced more than 11-fold by feeding 5% cholestyramine plus 0.1% lovastatin (the CL-diet) for 7 days but was severely suppressed by feeding 5% cholesterol or 0.01% AY-9944 (an inhibitor of 14-reductase) for the same period. The increase or decrease in the 14-reductase activity also parallels the same change in the cholesterol synthetic rate in hepatocytes from rats that had been fed either the CL-diet or 0.01% AY-9944. In vitro inhibition studies revealed that AY-9944 acts as a competitive inhibitor of the 14-reductase (Ki = 0.26 microM). A diurnal variation was observed for the 14-reductase with peak activity near the middle of the dark cycle (10 p.m.), which was abolished by administration of cycloheximide. With induced enzyme conditions 14-reductase has been further purified with chromatographic procedures to near homogeneity. Purified 14-reductase appears to be a M(r) = 70,000 protein that is composed of two equally-sized subunits having a M(r) = 38,000. All properties of the purified 14-reductase suggest that the solubilized enzyme is the principal 14-reductase of microsomes. Taken together, our results provide the first evidence in support of a previously unknown regulatory role for the 14-reductase in the overall cholesterol synthetic pathway.

Fungal growth within a silicone tissue expander: case report.

Fungal contamination of tissue expanders has not previously been reported. There are, however, reports of fungi in association with inflatable breast prostheses. Colonisation of a tissue expander with Aspergillus niger resulting in mechanical obstruction of the device is described. The possible modes of inoculation and survival of the organism within the expander envelope were studied including an investigation of the permeability to gases of the silicone expander envelope. Recommendations are made about prevention of this complication.

The effect of diagnosis and treatment delay on prognostic factors and survival in endometrial carcinoma.

OBJECTIVE: The aim of this study was to assess the association of diagnosis and treatment delay with established prognostic factors and survival. STUDY DESIGN: The study group comprised 181 consecutive patients with endometrial carcinoma diagnosed between 1970 and 1986, whose records contained details with regard to diagnosis delay; 174 of them also contained details with regard to treatment delay. RESULTS: The significant prognostic factors that we found, namely, age, clinical stage, grade, depth of myometrial invasion, and histologic type, are in line with those of other studies. However, no significant correlation was found between the duration of delay and these prognostic factors or with survival. CONCLUSION: We conclude that delay of diagnosis (< 1 year) and of treatment of < 4 months do not compromise survival of patients with endometrial cancer.

Blood-contacting biomaterials: bioengineering viewpoints.

The investigation of blood-contacting biomaterials is an important challenge and is relevant for an improvement in the clinical application of biomaterials. With the purpose of improved clinical treatment, bioengineering viewpoints of blood-contacting biomaterials cover the material options and selection, the utilization of materials, the development of materials with better properties, and processing characteristics, and the design of relevant evaluation procedures. The bioengineering objective remains that of achieving an enhanced understanding of the relationship between a biomaterial and the biological response.

Substrate-based inhibitors of lanosterol 14 alpha-methyl demethylase: II. Time-dependent enzyme inactivation by selected oxylanosterol analogs.

Selected 15-, 32-, and 15,32-substituted lanosterol analogs are shown here to display time-dependent inactivation and lanosterol 14 alpha-methyl demethylase. These molecules are competitive with respect to substrate and require NADPH and O2 in order to display time dependence, thus supporting the premise that they are mechanism-based inactivators. Structural features required for lanosterol demethylation by the lanosterol demethylase such as nuclear double bond location and availability of an abstractable 15 alpha-proton are also essential elements for time-dependent inactivation. 32-(S)-Vinyllanost-8-en-3 beta,32-diol is a potent time-dependent inactivator (Kinact/Ki = 0.36 min-1 microM-1), while the 32-(R)-vinyllanost-8-en-3 beta,32-diol functions solely as a competitive demethylase inhibitor. These results support the premise that stereoselective oxidation occurs during lanosterol demethylation and that the 32-pro-S proton is abstracted during the demethylation reaction.

Substrate-based inhibitors of lanosterol 14 alpha-methyl demethylase: I. Assessment of inhibitor structure-activity relationship and cholesterol biosynthesis inhibition properties.

A series of 15-, 32-, and 15,32-substituted lanost-8-en-3 beta-ols is described which function as inhibitors of cholesterol biosynthesis. These agents inhibit lanosterol 14 alpha-methyl demethylase activity as well as suppress HMG-CoA reduction activity in cultured cells. Several of these agents are extremely potent as both demethylase inhibitors and reductase suppressors, while others are more selective in their activities. Selected regio double bond isomers show preference for demethylase inhibition with the following order: delta 8 > delta 7 > delta 6 = unsaturated sterols. Comparisons also show that 4,4-dimethyl sterols are always more potent demethylase inhibitors and reductase suppressors than their 4,4-bisnomethyl counterparts. However, evaluation of an extensive oxylanosterol series leads us to conclude that demethylase inhibition and reductase suppression are not parallel in the same molecule. In addition, the oxylanosterols, but not the oxycholesterols, are able to disrupt coordinate regulation of HMG-CoA reductase from the LDL receptor. Thus, oxylanosterol treatment at levels which suppress reductase activity enhances LDL receptor activity. These results demonstrate that compounds can be made which (1) are selective reductase suppressors enabling dissection of the dual inhibitor nature of these compounds and (2) maximize reductase suppression and LDL receptor induction without demethylase inhibition which could lead to novel agents for serum cholesterol lowering.