Flow cytometric characterization of isolated porcine hepatocyte suspensions for liver support

The high yield hepatocyte isolation necessary for hybrid liver assist devices (LAD) unavoidably increases contamination by nonparenchymal cells and depresses hepatocyte viability and functions. We have developed a flow cytometric procedure that improves quality control of the isolations. Cells present in these preparations were labeled by immunofluorescent antibody staining against cytokeratin 8, 18 as well as vimentin to identify hepatocytes, fibroblasts, and endothelial cells. Antibody staining against albumin and carbamoylphosphate synthetase allowed assessment of levels of albumin and carbamoylphosphate synthetase based on the hepatocyte relative fluorescence intensity. Hepatocyte P450 enzyme activity was measured by its ability to convert 5,6-methoxycarbonylfluorescein, a nonfluorescent substrate, to an intracellular fluorescent product. Flow cytometric methods of cell type identification and cell function assessment are fast and accurate and can be applied to commercial cell production. They may also provide an avenue for the enrichment of otherwise heterogeneous hepatocyte suspensions with cells presenting the specific functions desired for an hybrid liver assist devices.     

Studies on the viability of the isolated vascularly perfused rat colon

The colon contains large numbers of endocrine cells. Insight into their physiological function is limited. This is due to the fact that no sufficient model of isolated endocrine colon cells is available. In the present study we introduce an isolated vascularly perfused colon model for in vitro studies. This model offers the advantage that it keeps the endocrine cells in their physiological orientation and environment. The gut mucosa is highly sensitive to ischemia. Therefore, a careful validation of its viability is crucial in gut organ preparations. This study demonstrates that, by utilizing an oxygenated vascular medium supplemented with 25% washed bovine erythrocytes, a perfusion of the colon is achieved for at least 1 h without obvious tissue injuries. During this time parameters such as perfusion pressure, venous lactate dehydrogenase release, glucose consumption, lactate output, oxygen consumption, perfusate loss by the preparation and morphology were analyzed. Dependent on stimulation, the endocrine L cells of the colon released glucagon-like peptide-I upon arterial perfusion of methacholine or gastrin-releasing peptide. In conclusion, a model for the isolated perfusion of the colon is introduced which is suitable for studies of endocrine colon cells.     

all-trans-retinoic acid preserves viability of fibroblasts and keratinocytes in full-thickness human skin and fibroblasts in isolated dermis in organ culture

Human dermal fibroblast and human epidermal keratinocyte survival was examined under various conditions in organ culture. Using cell recovery from organ-cultured tissue as the criterion, it was observed that no keratinocytes and few fibroblasts survived incubation for 10-12 days in serum-free basal medium containing a low level (0.15 mM) of extracellular Ca2+. Increasing the extracellular Ca2+ concentration to 1.4 mM or treating the tissue with 3 microM retinoic acid (RA) under low Ca2+ conditions resulted in increased keratinocyte and fibroblast survival; the two treatments together were more effective than either treatment alone. The same treatments preserved fibroblast survival when pieces of isolated dermal tissue were incubated in organ culture and also supported fibroblast survival in monolayer culture. These findings indicate that recovery of keratinocytes and fibroblasts from skin after maintenance in organ culture provides a simple but definitive measure of the viability of the major cellular elements present in the tissue. These findings suggest that RA treatment enhances survival of both fibroblasts and keratinocytes and that these effects of RA can be seen at physiological Ca2+ concentrations as well as at suboptimal levels of extracellular Ca2+. Finally, these results indicate that the dermis is a direct target of RA.     

Hepatobiliary effects of tertiary-butylhydroperoxide (tBOOH) in isolated rat hepatocyte couplets

CD40 ligand (CD40L) is involved in the T-cell-dependent regulation of B-cell growth and survival and can rescue normal germinal centre B cells and several types of malignant B cells from apoptosis in vitro. We have previously reported that serum of patients with chronic lymphocytic leukaemia contained elevated levels of biologically active soluble CD40L (sCD40L). Whether an augmented CD40L pathway exists in patients with other types of B-cell lymphoid malignancies and the source of native sCD40L in these patients is currently unknown. Using a sensitive ELISA assay, soluble CD40L (sCD40L) was detected in the sera of both healthy individuals and patients with haematological malignancies; however, its level was significantly elevated only in patients with B-cell lymphomas (P<0.0001). Several types of malignant B cells coexpressed CD40 and CD40L proteins, and CD40L mRNA was detected in purified resting malignant B cells. The dual expression of CD40 and CD40L in B cells and the presence of native sCD40L in human serum suggest that a direct T-B-cell contact may not be required for CD40L delivery to B cells. This data raises the possibility that an autocrine cytokine loop involving CD40L may contribute to the growth regulation of benign and malignant B cells in vivo.     

Investigation of oxidant stress and vasodepression to glyceryl trinitrate in the obese Zucker rat in vivo

OBJECTIVE: To describe risk factors and explore mechanisms of ischemic strokes after general surgery. BACKGROUND: Strokes follow general surgery in about 0.08% to 2.9% of cases. Patients with previous cerebrovascular disease, atrial fibrillation, hypertension, advanced age, or atherosclerosis were found to have an increased risk. Knowledge of factors involved may guide physicians in determining the overall risk of surgery. METHODS: This case-control study was performed in a referral center. A total of 61 patients identified through a computerized database with ischemic strokes after surgical procedures-excluding heart, brain, vessels, or neck-between July 1986 and July 1996 were studied. Procedures included 11 urogenital, 16 gastrointestinal, 17 orthopedic, 12 pulmonary, and 5 other. A total of 122 randomly selected controls were matched for age, sex, procedure, and year of procedure. Main outcome measures included arterial territory, timing, risk factors, and perioperative events. Differences were expressed as adjusted odds ratios (AOR) with 95% confidence limits (CL), using multivariate conditional logistic analyses for matched case-control design. RESULTS: Arterial territory included 37 middle cerebral artery, 11 posterior circulation, 7 borderzone, and 6 multiple. Median procedure to stroke interval was 2 days (range, 0 to 16); 10 patients had intraoperative strokes. Three major risk factors emerged: previous cerebrovascular disease (AOR 12.57, 95% CL 2.14/73.70), chronic obstructive pulmonary disease (COPD) (7.51, 1.87/30.12), and peripheral vascular disease (PVD) (5.35, 1.25/22.94). After adding stroke-related factors, PVD (14.70, 2.01/107.71) and COPD (10.04, 1.90/53.14) remained the strongest variables; blood pressure (1.05, 1.01/1.10) and urea (1.04, 1.01/1.07) contributed slightly. Hypotension did not contribute. Four patients (6.6%) and no controls had diffuse intravascular coagulation (p = 0.01). Four stroke patients had myocardial infarction (6.6% versus 0%; p = 0.01). CONCLUSIONS: Ischemic strokes after general surgery most commonly occur after an asymptomatic interval. Previous cerebrovascular disease, COPD, and PVD greatly increase the risk. Hypotension rarely accounts for postoperative strokes. Major comorbidity of the patient at risk seems more important than complicating events during surgery.     

Ethionine toxicity in vitro: the correlation of data from rat hepatocyte suspensions and monolayers with in vivo observations

The hepato-steatogenic compound ethionine has been used to investigate the correlations between in vivo and in vitro toxicity data. The aim was to find a suitable model of toxicity in hepatocyte suspensions or monolayers in vitro, which could predict the known toxicity of ethionine in vivo and which could be implemented in screening compounds of unknown toxicity. Thus a variety of markers of cytotoxicity, metabolic competence and liver-specific functions were investigated in rat hepatocyte suspensions and monolayers and compared with in vivo data in the rat. The following markers were measured in the appropriate system: (1) Neutral red uptake; 3-(4,5 dimethyl)thiazol-2-yl,-2,5-diphenyl tetrazolium bromide (MTT) reduction; lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) leakage (cytotoxicity). (2) ATP levels, protein synthesis and glutathione (GSH) levels (metabolic competence). (3) Urea and triglyceride synthesis and beta-oxidation (liver specific functions). Ethionine (0-30 mM) did not affect the markers of direct cytotoxicity, except neutral red uptake, which was reduced by 18 and 30 mM ethionine after 20 h in culture. ATP and GSH depletion occurred in hepatocyte suspensions at the highest concentrations of ethionine (20 and 30 mM) after 1 h. In monolayers, GSH levels were reduced after 4 h, but not 20 h. Urea synthesis was increased in hepatocyte suspensions from 1 to 3 h by 10-30 mM ethionine and reduced after 20 h in cultured hepatocytes (18-30 mM). Protein synthesis was reduced and beta-oxidation was increased in ethionine-treated hepatocyte suspensions. Unfortunately, there was no measurable effect on triglyceride accumulation within cells (the major biochemical change in vivo) in either system. Ethionine treated hepatocytes in suspension showed the same rate of triglyceride synthesis and transportation out of cells as control cells. Thus, hepatocyte suspensions were able to mimic the early biochemical effects of ethionine in vivo (ATP and GSH depletion, inhibition of protein synthesis) and some effects on urea synthesis, but monolayer cultures appeared to be less sensitive to the toxicity of ethionine. However, neither in vitro system was able to model the effects of ethionine on the accumulation of triglycerides in vivo.     

The effect of hepatocyte enlargement on the hemodynamic characteristics of the isolated perfused rat liver preparation

The influence of hepatocyte enlargement on intrahepatic hemodynamics was assessed in the isolated perfused rat liver preparation (IPRL) using two experimental models: hypotonic liver cell swelling and phenobarbitone-induced hepatocyte hypertrophy. The analysis of pressure-flow data obtained from the portal vascular bed over a flow range of 0 to 70 mL/min in the presence of a maximally-effective concentration of the vasodilator agent papaverine hydrochloride (6 x 10(-4) mol/L) enabled the calculation of P0, an estimate of the pressure required to passively distend the intrahepatic vasculature, and Gmax, the maximal portal vascular conductance. By comparison with an isotonic perfusion medium (Krebs-Henseleit buffer [KH] containing 2.5% bovine serum albumin [BSA]), perfusion with a hypotonic medium induced a significant increase in mean hepatocyte cross-sectional area (H(A)) (590 +/- 21 vs. 324 +/- 23 microm(-2), p < .05), a fall in Gmax (0.39 +/- 0.08 vs. 2.02 +/- 0.18 mL/min/g/mm hg, P < .001), and an increase in P0 (2.96 +/- 0.38 vs. 1.58 +/- 0.07 mm hg, P < .001). Phenobarbitone administered in drinking water (0.5 g/L) over a period of 60 days also induced a significant degree of hepatocyte enlargement (HA, 510 +/- 29 microm2, P < .05). On day 7, portal pressure measured in vivo in this group was significantly elevated compared with untreated controls (10.5 +/- 0.3 vs. 8.4 +/- 0.2 mm hg, P < .001), while in the IPRL Gmax was reduced (0.48 +/- 0.01 mL/min/g/mm hg, P < .001), and P0 was increased (2.23 +/- 0.17 mm hg, P < .05). However, with continued phenobarbitone treatment portal pressure, Gmax and P0 returned toward control values. The results confirm that hepatocyte enlargement is associated with a significant disturbance of intrahepatic hemodynamics but also that some adaptation occurs if hepatocyte enlargement is sustained over a prolonged period of time.     

Recovery by ascorbate of impaired nitric oxide-dependent relaxation resulting from oxidant stress in rat aorta

1. In this study we investigated the ability of ascorbate to protect nitric oxide from destruction by superoxide anion. 2. Ascorbate produced concentration-dependent relaxation of rings of rat aorta, comprising two components: the first, seen at 1-300 microM, reached a maximum of 45.3+/-2.8%, and was abolished by endothelial removal or treatment with L-NAME (100 microM), demonstrating involvement of nitric oxide. The second occurred at concentrations of 1 mM and above and was associated with falls in the pH of the bathing fluid. 3. Pretreatment with ascorbate at concentrations up to 3 mM had no effect on the relaxation to acetylcholine (10 nM-10 microM) on endothelium-containing rings or adenosine (0.1 microM-3 mM) on endothelium-denuded rings. 4. An oxidant stress was applied to aortic rings, comprising inhibition of endogenous Cu/Zn superoxide dismutase by diethyldithiocarbamate (0.1 mM) followed by generation of superoxide anion by hypoxanthine (0.1 mM/xanthine oxidase (16 u ml(-1)). This reduced maximal acetylcholine-induced relaxation from 96.7+/-1.3% to 42.4+/-3.5% (P<0.001). Treatment with ascorbate (30 microM-3 mM) reversed this blockade in a concentration-dependent manner. 5. Our findings show that ascorbate has the ability to protect nitric oxide from destruction by superoxide anion. This action is seen with ascorbate at levels normally present in plasma, suggesting that this antioxidant may exert a tonic protective effect on nitric oxide within the vasculature.     

Glutathione disulfide as an index of oxidative stress during postischemic reperfusion in isolated rat hearts

Junctional sequences of immunoglobulin (Ig)/T-cell receptor (TCR) gene rearrangements are used as patient-specific PCR targets for the detection of minimal residual disease (MRD) in acute lymphoblastic leukaemias (ALLs). Clonal evolution of gene rearrangements is a major pitfall of this strategy. Using high-resolution PCR-based analyses (including denaturing gel electrophoresis and single-stranded conformation polymorphism (SSCP)) we have compared Ig/TCR gene rearrangements at presentation and relapse in a series of ALLs. These methods allow an unambigous comparison of rearrangements taking into account junctional size and nucleotide sequence information and allow a precise assessment of the clonal evolution. V gamma-J gamma and V delta 1-J delta 1 rearrangements were analysed in 12 T-ALLs. VH-JH, V gamma-J gamma, V delta 2-D delta 3 and, in selected cases, DH-JH rearrangements were studied in 14 B-lineage ALLs. Clonal evolution, regarding major rearrangements, occurs for at least one of these loci in 2/12 T-ALLs and in 5/14 B-lineage ALLs. Clonal evolution is more marked for minor rearrangements than for major ones. As shown using SSCP analysis, rearrangements observed at relapse are sometimes found in minor clones at presentation which are therefore selected in vivo by a proliferative advantage. These data, as well as those from the available literature, suggest the use of at least two patient-specific probes to detect MRD in ALLs. A general strategy including selected Ig/TCR rearrangements and chromosomal abnormalities as PCR targets is proposed.     

Adenovirus-mediated catalase gene transfer reduces oxidant stress in human, porcine and rat pancreatic islets

Susceptibility of pancreatic islets to oxidant stress may affect islet viability and contribute to primary non function of allo- or xenogenic grafts. We investigated the influence of overexpression of catalase (CAT) on the viability of human, porcine and rat islets, as well as INS-1 beta-cell line. Islets were transfected with a replication-deficient adenovirus vector containing human CAT cDNA under the control of the adenovirus major late promoter (AdCAT) or a vector containing no foreign gene (AdNull) and used as a control. Oxidant stress was induced 48 h later by xanthine oxidase-hypoxanthine (XO 25 mU/ml, HX 0.5 mmol/l) or hydrogen peroxide (100 or 250 micromol/l). Islet cell viability was assessed 72 h after CAT transfer by 4-[3-(4-Idophenyl)-2-(4 nitrophenyl)-2H-5-tetrazolio]-1,2,benzene disulphonate (WST-1) test. Baseline catalase activity was three to fourfold lower in porcine than in human islets. CAT activity was reproducibly increased 2.5- to 7-fold in AdCAT infected islets, at least for 13 days. Overall, AdCAT conferred on human and pig islets a protection of 26.1 +/- 6.1 and 21.2 +/- 9.8% on XOHX injury and 35.4 +/- 4.2 and 57.9 +/- 10.5% on H2O2 stress. Similarly, rat islet cells and INS-1 cells were protected on XOHX stress by 17.8 +/- 2.3 and 30.8 +/- 8.7%, respectively. AdNull had no effect. Basal and stimulated insulin secretion was preserved in AdCAT-transfected human islets despite a XOHX challenge. This study validates adenovirus-mediated catalase gene transfer as a realistic approach to reduce non specific inflammation effects on human or porcine islet grafts. Moreover the relevance of defense mechanisms, previously suggested in human islets, is here illustrated in porcine islets.     

Protein secretion in suspensions of isolated rat hepatocytes: no influence of acute ethanol administration

The effect of ethanol on the secretion of proteins was studied in hepatocytes isolated from 24-h fasted rats and from fed rats. Hepatocytes were isolated after collagenase disruption of the liver and incubated in a standard medium containing amino acids, bovine albumin, glucose, penicillin and streptomycin in HEPES buffer. Cell viability was determined by urea production and trypan blue exclusion. When studying protein export, a model had to be chosen in which the labeling is accomplished before the addition of the test agents. Cells were incubated with [3H]valine for 2.5 and 7.5 min followed by a 15-mM valine chase and the incubates were adjusted to final concentrations of ethanol of 50 mM, 100 mM, colchicine 5-50 microM or cycloheximide 18 microM. Cells and media were harvested at various times, and counts incorporated into medium and cell protein were determined. Cycloheximide inhibited protein synthesis by 99%, decreased protein secretion by 10-20%, but did not further inihibit protein labeling when given after the chase confirming the chase's effectiveness. Colchicine inhibited protein release by 27-54% depending on the dose. With control cells labeled protein and specifically albumin appeared in the medium 20 min from the start of the pulse and this release of protein was not inhibited by 50 mM or 100 mM ethanol incubated with cells from the same animal whether the donor has been fed or fasted. The values for the ethanol-treated cells ranged from 94.0 to 113% of the control values from 30 to 120 min after the addition of the pulse. Lactate levels were markedly elevated, and urea synthesis decreased in the presence of either 50 mM EtOH or 100 mM EtOH. Thus using a method that can distinguish the effect of ethanol on synthesis from secretion, it is concluded that acute exposure to EtOH does not interfere with protein secretion.     

Oxidative stress response induced in rat primary hepatocyte monolayers by mechanical removal of adherent cells

In a previous study, we generated a mutant of dHGF (deleted variant of hepatocyte growth factor), termed #2, with higher specific activity than dHGF in assays of mitogenic activity on rat hepatocytes and America opossum kidney epithelial cells (OK). In the present study, we examine in vivo hepatotropic and renotropic activities of #2 and its distribution to target tissues, liver and kidney. Administration of #2 to normal rats significantly increased serum levels of total protein, albumin, free-cholesterol, and HDL-cholesterol and liver weight in a dose-dependent manner. Analysis of these parameters suggests that #2 is more potent than dHGF as a hepatotropic factor in vivo. In addition, #2 reduced mortality of mercuric chloride-administered mice and the effect was stronger than that of dHGF. When injected to mice, a larger amount of #2 than dHGF was rapidly distributed to the liver. Sixty minutes after injection, the concentrations of #2 in plasma, liver, and kidney were higher than those of dHGF. These distribution properties and the higher mitogenic activity in vitro may explain why #2 exerts more potent in vivo biological activity than dHGF.     

Is hemin responsible for the susceptibility of Plasmodia to oxidant stress?

Based on the unusually high and stage-dependent susceptibility of Plasmodia to oxidant stress it has been proposed that during parasite development, increasing levels of redox-active forms of iron are gradually released. The purpose of this study was to examine this proposal by using an assay monitoring the levels of available forms of iron for redox reactions. Ascorbate-driven and iron-mediated degradation of adventitious DNA served as the basis for this functional assay. Incubation of DNA with lysate from infected RBC caused massive degradation, which was dose, time- and parasite-stage dependent. In contrast, lysate from non-infected RBC did not induce DNA degradation. Likewise, lysate only from infected RBC enhanced the aerobic oxidation of ascorbate. These effects on both reaction, DNA degradation and ascorbate oxidation, could be reconstructed with hemin, instead of lysate. Also, chelators exerted similar effects on both reactions. The results suggest that increased levels of redox-active forms of iron are liberated during parasite development. We propose that hemin or hemin-like structures are the appropriate candidates which could catalyze oxidative stress and deregulate the delicate redox balance of the host-parasite system.     

Detection of myocardial viability by stress echocardiography

Dobutamine echocardiography has gradually acquired a place among the currently available techniques to evaluate myocardial viability. It is a very efficient technique, easily accessible and inexpensive. It detects preservation of the myocyte contractile apparatus, which unlike other tests best assesses myocardial viability. Its value in stunned and hibernating myocardium is herein described.     

Markers of platelet activation and oxidant stress in atherothrombotic disease

Several new approaches to the study of platelet activation have been developed. Logically, these should be combined with novel indices of coagulant function (60,61) to select rational targets for antithrombotic drugs. They may also be invaluable in dose-finding, which has been a particular weakness in this area of drug development (62,63). While activation of platelets and the coagulation cascade are virtually simultaneous events, markers of the atherosclerosis are also artificially segregated from those of the complicating thrombotic process. Oxidant stress has been implicated in both platelet activation (64) and atherogenesis (65), yet our ability to study this system has been so constrained that we are unsure of appropriate doses of antioxidant vitamins. Novel approaches to this problem promise the ability to study oxidative modification of proteins (46,66,67), lipids (57) and DNA (45,68) in clinical studies.     

Analysis of air pollution particulate-mediated oxidant stress in alveolar macrophages

Adverse health effects of urban air pollution particulates may be attributable to particle-mediated oxidant stress and inflammation. Intracellular oxidant production in normal hamster alveolar macrophages (AMs) was measured upon exposure to concentrated ambient particulates (CAPs), residual oil fly ash (ROFA), and their water-soluble and particulate fractions. ROFA and CAPs caused increases in dichlorofluorescin (DCFH) oxidation, a fluorescent measure of intracellular reactive oxygen species (ROS) production, comparable to the positive control, phorbol myristate acetate (PMA). The water-soluble component of both CAPs and ROFA (CAPs, S and ROFA, S) significantly increased AM oxidant production over negative control. CAPs samples and components showed substantial day-to-day variability in their oxidant effects. Metal chelation by desferrioxamine (DF, 1 mM) caused significant inhibition of particulate-induced AM oxidant production. ROFA exposure resulted in increased macrophage inflammatory protein-2 (MIP-2) message in AMs and in increased tumor necrosis factor alpha (TNF-alpha) production by the monocyte-macrophage cell line, RAW 264.7. TNF-alpha production was inhibitable by the antioxidant N-acetylcysteine (NAC). The data suggest that metal components adsorbed to urban air pollution particulates can significantly contribute to particulate ability to cause oxidant stress and cytokine production in AMs.     

Improvement of perfusion-flow in the isolated rat liver, under the influence of streptase

Accumulation of radioactive material in the isolated rat liver was noted when 125I-fibrinogen was added to the perfusing medium. Owing to an enhanced production of fibrinogen and to a diminished fibrinolysis, fibrin deposits were found to occur in the capillaries of fhe isolated liver. This would cause a decrease of hepatic flow and an impairment of the nutrition of this organ which leads to subsequent necrosis. Administration of streptase removed the fibrin deposits and restored the perfusive flow in the isolated liver.