[Research progress of ischemic hepatitis].

Ischemic hepatitis, also known as hypoxic hepatitis or shock liver, refers to liver cell damage without any known cause of acute hepatitis, and is characterized by transient elevation of transaminase levels (20 times higher than normal value).The incidence of the disease is about 2.5% to 10%, and the hospital mortality rate is greater than 50%. Current research suggests that there are many risk factors for the disease, including systemic hypotension, low cardiac output, sepsis and respiratory distress, but eventually it will manifest as hepatocyte dysfunction. Unfortunately, the mortality rate related with hypotension is high, and the key to treatment is to correct hemodynamic disorders. This article reviews the research progress in the etiology, mechanism and clinical manifestations of ischemic hepatitis.

Vascular endothelial growth factor KDR receptor signaling potentiates tumor necrosis factor-induced tissue factor expression in endothelial cells.

Vascular endothelial growth factor (VEGF) and tumor necrosis factor-alpha (TNF-alpha) have been shown to synergistically increase tissue factor (TF) expression in endothelial cells; however, the role of the VEGF receptors (KDR, Flt-1, and neuropilin) in this process is unclear. Here we report that VEGF binding to the KDR receptor is necessary and sufficient for the potentiation of TNF-induced TF expression in human umbilical vein endothelial cells. TF expression was evaluated by Western blot analysis and fluorescence-activated cell sorting. In the absence of TNF-alpha, wild-type VEGF- or KDR receptor-selective variants induced an approximate 7-fold increase in total TF expression. Treatment with TNF alone produced an approximate 110-fold increase in total TF expression, whereas coincubation of TNF-alpha with wild-type VEGF- or KDR-selective variants resulted in an approximate 250-fold increase in TF expression. VEGF lacking the heparin binding domain was also able to potentiate TF expression, indicating that heparin-sulfate proteoglycan or neuropilin binding is not required for TF up-regulation. Neither placental growth factor nor an Flt-1-selective variant was capable of inducing TF expression in the presence or absence of TNF. Inhibition of protein-tyrosine kinase or protein kinase C activity significantly blocked the TNF/VEGF potentiation of TF up-regulation, whereas phorbol 12-myristate 13-acetate, a protein kinase C activator, increased TF expression. These data demonstrate that KDR receptor signaling governs both VEGF-induced TF expression and the potentiation of TNF-induced up-regulation of TF.

Vascular endothelial growth factor governs endothelial nitric-oxide synthase expression via a KDR/Flk-1 receptor and a protein kinase C signaling pathway.

The mechanism by which vascular endothelial growth factor (VEGF) regulates endothelial nitric-oxide synthase (eNOS) expression is presently unclear. Here we report that VEGF treatment of bovine adrenal cortex endothelial cells resulted in a 5-fold increase in both eNOS protein and activity. Endothelial NOS expression was maximal following 2 days of constant VEGF exposure (500 pM) and declined to base-line levels by day 5. The elevated eNOS protein level was sustained over the time course if VEGF was co-incubated with L-N(G)-nitroarginine methyl ester, a competitive eNOS inhibitor. Addition of S-nitroso-N-acetylpenicillamine, a nitric oxide donor, prevented VEGF-induced eNOS up-regulation. These data suggest that nitric oxide participates in a negative feedback mechanism regulating eNOS expression. Various approaches were used to investigate the role of the two high affinity VEGF receptors in eNOS up-regulation. A KDR receptor-selective mutant increased eNOS expression, whereas an Flt-1 receptor-selective mutant did not. Furthermore, VEGF treatment increased eNOS expression in a KDR but not in an Flt-1 receptor-transfected porcine aorta endothelial cell line. SU1498, a selective inhibitor of the KDR receptor tyrosine kinase, blocked eNOS up-regulation, thus providing further evidence that the KDR receptor signals for eNOS up-regulation. Finally, treatment of adrenal cortex endothelial cells with VEGF or phorbol ester resulted in protein kinase C activation and elevated eNOS expression, whereas inhibition of protein kinase C with isoform-specific inhibitors abolished VEGF-induced eNOS up-regulation. Taken together, these data demonstrate that VEGF increases eNOS expression via activation of the KDR receptor tyrosine kinase and a downstream protein kinase C signaling pathway.

Hepatocyte growth factor inhibits amiloride-sensitive Na(+) channel function in cystic fibrosis airway epithelium in vitro.

Cystic fibrosis (CF) airway epithelial cells have a reduced cAMP-dependent Cl(-)conductance channel (CFTR) function but an increased level of amiloride-sensitive Na(+)channel (ENaC) activity. Recently, expression of the alpha -subunit of the ENaC protein complex was shown to be down-regulated by activation of the extracellular signal-regulated protein kinase (ERK) pathway. In the present study we have examined the actions of a potent regulator of the ERK pathway, recombinant human hepatocyte growth factor (rhHGF), on the function of ENaC in confluent, polarized monolayers of both primary cultures of CF airway cells and an SV40-transformed CF nasal epithelial cell line (JME CF/15). Treatment of JME/CF 15 cells with rhHGF at concentrations of 100 ng/ml and above was found to dramatically decrease the activity of amiloride-sensitive Na(+)transport. This effect required basolateral exposure of the cytokine. Addition of 100 ng/ml rhHGF to JME/CF 15 cells decreased I(eq)with a t(1/2)of;18 h, with a maximal inhibition of;90% by 36 h. By 48 h, stimulation with rhHGF induced a down-regulation of its receptor, c-met, expressed in these cells. The decrease in I(eq)of JME/CF 15 monolayers was not immediately reversed upon removal of rhHGF. Treatment with rhHGF did not appear to affect monolayer resistances nor Cl(-)currents induced by mediators such as isoproterenol, histamine or bradykinin. Studies with primary cultures of CF airway cell sheets demonstrated comparable sensitivity and time-course properties for the inhibition of amiloride-sensitive currents following rhHGF addition. These observations are consistent with the possible application of an extracellular signalling molecule, such as the cytokine HGF, to reduce the abnormally high activity of amiloride-sensitive Na(+)ion channels observed in CF airway cells.

Homologous up-regulation of KDR/Flk-1 receptor expression by vascular endothelial growth factor in vitro.

We investigated the possibility that vascular endothelial growth factor (VEGF) treatment could regulate KDR/Flk-1 receptor expression in endothelial cells. Bovine adrenal cortex endothelial cells were incubated with 200 pM rhVEGF165 for 0-7 days. Western blot analysis showed a 3-5-fold increase in total KDR protein following 4-day VEGF treatment. Scatchard analysis revealed that VEGF induced a 2-3-fold increase in high affinity receptor number (5.0 x 10(4)/cell versus 2. 4 x 10(4)/cell) without significantly affecting receptor binding affinity (Kd 76 pM versus 72 pM). Quantitative polymerase chain reaction analysis demonstrated a 3-fold increase in KDR mRNA levels following VEGF exposure. VEGF-induced KDR expression primarily occurred at the transcriptional level as demonstrated by a luciferase reporter assay system. Receptor selective mutants with wild-type KDR binding and decreased Flt-1 binding also induced KDR up-regulation; in contrast, mutants with decreased KDR binding and wild-type Flt-1 binding did not, suggesting that KDR receptor signaling mediated the increase in KDR expression. Inhibition of tyrosine kinase, Src tyrosine kinase, protein kinase C, and mitogen-activated protein kinase activities all blocked VEGF-induced KDR up-regulation. Finally, co-incubation of nitric-oxide synthase inhibitors with VEGF had no significant effect on KDR expression, but 100 microM sodium nitroprusside, a NO donor, significantly inhibited VEGF-induced KDR up-regulation, indicating that NO negatively regulates KDR expression. In conclusion, our data demonstrate that VEGF binding to the KDR receptor tyrosine kinase results in an increase in KDR receptor gene transcription and protein expression. Thus, KDR up-regulation induced by VEGF may represent an important positive feedback mechanism for VEGF action in tumor and ischemia-induced angiogenesis.

Campylobacter jejuni lipopolysaccharides in Guillain-Barré syndrome: molecular mimicry and host susceptibility.

OBJECTIVE: This study was designed to determine if the presence of specific ganglioside-like moieties in Campylobacter lipopolysaccharides (LPSs) is related to the development of Guillain-Barré syndrome (GBS), and to discover how frequently such moieties, including GM1, are present in these LPSs. METHODS: We studied Campylobacter isolates and sera from seven patients with GBS (five acute motor axonal neuropathy, one acute inflammatory demyelinating polyneuropathy, and one Fisher's syndrome), and compared them with similar specimens from patients with Campylobacter enteritis alone. RESULTS: All GBS patients had antiganglioside antibodies. Anti-GM1 and anti-GD1a titers were significantly elevated in post-Campylobacter GBS, both axonal and demyelinating, compared with normal control subjects or those with uncomplicated Campylobacter diarrhea. Campylobacter isolated from patients with GBS and with enteritis alone had similar ganglioside-like moieties. CONCLUSIONS: These results indicate that patients who develop GBS respond differently to the ganglioside-like epitopes on Campylobacter than do non-GBS diarrhea patients. Our findings support a role for host susceptibility as a determinant for the outcome following Campylobacter infection. These findings have important implications for the development of vaccines against Campylobacter jejuni.

Hepatocyte growth factor stimulates the differentiation of human tracheal epithelia in vitro.

Hepatocyte growth factor (HGF) can influence epithelial cell growth and differentiation. We examined the actions of recombinant human HGF (rhHGF) on the differentiation of human primary tracheal epithelial (HTE) cells cultured in vitro for up to 96 h. Basolateral, but not apical, treatment of confluent HTE cell sheets for 48 h with rhHGF led to increases in cell height, cell volume, cilia, and total protein content. Basolateral rhHGF treatment produced a decrease in HGF receptor (c-met) expression but had no effect on c-met mRNA levels. HTE cell sheets treated with rhHGF for 48 h showed a significant increase in mediator-induced Cl- secretion and a decrease in amiloride-sensitive sodium absorption. No effect on transepithelial resistance was observed with rhHGF treatment. The enhancement of short-circuit responses by basolateral rhHGF was dose dependent. Our results demonstrate that rhHGF has hypertrophic actions on, and can influence the differentiation of, human airway epithelia in vitro, presumably through the activation of c-met at the basolateral surface of these cells.

PCR-based restriction fragment length polymorphism (RFLP) analysis and serotyping of Campylobacter jejuni isolates from diarrheic patients in China and Japan.

A molecular typing approach for Campylobacter jejuni was applied with restriction fragment length polymorphism (RFLP) analysis of a 702-bp PCR-amplified portion of the flagellin-A (flaA) gene. We analyzed a total of 179 strains, including 69 independent clinical isolates from diarrheic patients in Japan, 85 isolates in China, and 25 heat-stable (HS) serotype strains by Penner and Hennessy (1980) J. Clin. Microbiol. 12, 732-737). Six AfaI, seven MboI, and five HaeIII RFLPs were found in the 702-bp flaA segment from the 179 strains. Using a combination of these three enzymes, 25 separate RFLP groups were recognized. While 59 of 154 (38.3%) strains obtained in Japan and China were nontypeable by the HS antigenic scheme, all but two of 154 (98.7%) could be typed by RFLP typing. All 11 isolates of HS-19 strains, which are frequently isolated from Guillain-Barré syndrome (GBS) patients, showed an identical RFLP pattern (Cj-1), and Cj-1 consisted only of HS-19 strains. This suggests that the HS-19:Cj-1 strain is distinct among C. jejuni strains. This molecular typing method provides a rapid and reliable typing scheme for epidemiological studies of C. jejuni, and may also be useful for the analysis of C. jejuni subtypes from GBS patients.

Kinetics of retroviral production from the amphotropic ΨCRIP murine producer cell line.

Rapidly expanding development and practice of gene therapy requires the availability of large quantities of high titer retroviral supernatants. One way to achieve high retroviral titers is through improved understanding of the kinetics of retroviral production and decay, and the subsequent development of improved cell culture methods. In the present study we investigated the effects of different operational modes on the retroviral production of the NIH 3T3 fibroblast derived amphotropic murine retroviral producing cell line pMFG/ΨCRIP. Semi-continuous culture (exchange of 50% of medium volume daily) was found to promote cell growth and enhance retroviral production. The rapid medium exchange resulted in significantly larger amounts of high titer supernatants and an extended production phase as compared to the batch control cultures. The specific viral productivity of the pMFG/ΨCRIP cells was in the range of 10 to 40 infectious viruses produced per thousand producer cells per day. The CV-1 African Green Monkey kidney cell line was used as the infection target. Lowering the serum level form 20% to 10% improved retroviral production slightly. However, at lower serum levels (1%, 5% and 10% (v/v)) growth of the producer cell line, and thus retroviral production, was directly proportional to the serum level. The half-life of the virus at 37°C was found to be 5.5 hours. Promoting the growth of producer cell lines can improve retroviral vectors titers and viral production. High cell density systems that allow for rapid cell growth and waste product removal are likely to be used to generate high-titer retroviral supernatants.

Role of CFTR in chloride secretion across human tracheal epithelium.

We have tested two hypotheses: 1) the cystic fibrosis transmembrane conductance regulator (CFTR) represents the predominant Cl conductance in the apical membrane of human tracheal epithelium, and 2) CFTR in this tissue is close to maximally activated under baseline conditions. In support of the first hypothesis, we found 1) when the level of differentiation of cultures was varied by varying the culture conditions, there was a significant positive correlation between the levels of CFTR and the magnitude of mediator-induced Cl secretion. 2) Amiloride-insensitive baseline short-circuit current (Isc) and mediator-induced increases in Isc were inhibited by diphenylamine-2-carboxylic acid (DPAC) but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a pharmacology consistent with passage of apical membrane Cl current through CFTR; Ca-activated Cl channels are inhibited by DIDS but not by DPAC. 3) Raising temperature from 22 degrees to 37 degrees C increased 125I efflux, and this increase was inhibited by DPAC and blockers of protein kinase A, but not by DIDS or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester. In support of the second hypothesis, we have earlier shown [M. Yamaya, W.E. Finkbeiner, S.Y. Chun, and J.H. Widdicombe. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L713-L724, 1992] that adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents are essentially without effect on Isc across primary cultures of human tracheal epithelium. Here, we further show that these agents are also usually without effect on 125I efflux; the mean increase in efflux in response to elevating cAMP was approximately 20% that of raising temperature from 22 degrees to 37 degrees C.

Effects of lovastatin on trafficking of cystic fibrosis transmembrane conductance regulator in human tracheal epithelium.

Genetic defects in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel, cause cystic fibrosis. Most defective forms of CFTR show improper intracellular trafficking. Because isoprenylated, small GTP-binding proteins are involved in the vesicular trafficking of other integral membrane proteins, we have investigated the role of isoprenylation in the trafficking of CFTR to the apical membranes of primary cultures of human airway epithelium and of Calu-3 cells, a human lung carcinoma cell line. CFTR function was measured as short circuit current, 125I efflux, and conductance of cell sheets with permeabilized basolateral membranes. Lovastatin, an inhibitor of isoprenyl lipid biosynthesis, markedly inhibited all measures of CFTR function. The lovastatin-induced declines in CFTR function were corrected by the simultaneous addition of mevalonate or the isoprenyl lipids geranylgeranyl and farnesyl but not cholesterol. Lovastatin reduced total cellular CFTR as assessed by immunoprecipitation. Mevalonate or isoprenyl lipids protected CFTR levels from the actions of lovastatin. Together, these results suggest a role for isoprenyl lipids, presumably through the actions of small GTP-binding proteins, in the trafficking of CFTR to the apical membrane of human airway epithelium.

A method for measuring Cl efflux from dispersed cells of airway epithelium.

This paper describes a method for measuring the increase in halide permeability of isolated airway epithelial cells induced by adenosine 3',5'-cyclic monophosphate (cAMP). Suspensions of isolated cells, known to contain the cystic fibrosis transmembrane conductance regulator (CFTR), were placed in the upper part of a Swinnex filter holder containing a filter with pores of 0.65 micron diameter. Medium was perfused over the cells at room temperature and collected at minute intervals following its passage through the filter. Experiments were performed on Calu-3 and T84 cells (human lung and colonic epithelial cell lines), primary cultures of dog and human tracheal epithelium, and Swiss 3T3 fibroblasts stably transfected with CFTR. In all cell types, addition of agents that elevate cAMP led to increases in the rates of loss of 36Cl and 125I. However, in human tracheal epithelial cells, warming the medium from room temperature to 37 degrees C was a more effective way of stimulating tracer efflux. Increases in efflux in response to either temperature or cAMP-elevating agents were inhibited by diphenylamine-2-carboxylate, a blocker of CFTR. Reproducible increases in tracer efflux were seen with as few as 10(6) cells. Cells that had been trypsinized off their culture dishes responded better than cells that had been scraped off, although treatment of scraped cells with trypsin enhanced their responsiveness to cAMP-elevating agents. Cystic fibrosis is characterized by the lack of a cAMP-activated Cl conductance in the apical membrane of airway epithlia.(ABSTRACT TRUNCATED AT 250 WORDS)

Chloride secretion and function of serous and mucous cells of human airway glands.

Cells from the acini of human tracheal glands were grown in culture to produce confluent cell sheets of mucous or mixed seromucous phenotype. Levels of mediator-induced Cl secretion in mucous cells were 2-18% those of seromucous cells. Levels of the cystic fibrosis transmembrane conductance regulator (an apical membrane Cl channel) were also much less in mucous than in seromucous cells. These results suggest that serous cells are more important than mucous cells in providing the fluid component of gland secretions.

Calu-3: a human airway epithelial cell line that shows cAMP-dependent Cl- secretion.

Of 12 cell lines derived from human lung cancers, only Calu-3 cells showed high transepithelial resistance (Rte) and increases in short-circuit current (Isc) in response to mediators. Calu-3 cells formed polarized monolayers with tight junctions and Rte of approximately 100 omega.cm2. Baseline Isc was approximately 35 microA/cm2 and was increased by approximately 75 microA/cm2 on elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) by isoproterenol. Flux studies showed that the increase in Isc was due to Cl- secretion. Forskolin and permeant analogues of cAMP also increased Isc. Consistent with the presence of cAMP-dependent Cl- secretion, immunoprecipitation demonstrated the presence of the cystic fibrosis transmembrane conductance regulator (CFTR). Bradykinin, methacholine, trypsin, and histamine all transiently (15-30 s) elevated Isc, probably by increasing intracellular Ca concentration. Experiments in which the basolateral membrane was permeabilized with nystatin indicated that CFTR was substantially activated under baseline conditions and that Ca-activated Cl- channels were absent from the apical membrane. We anticipate that Calu-3 cells will prove useful in the study of Cl- secretion and other functions of human airway epithelial cells.

Hybridoma cells in a protein-free medium within a composite gel perfusion bioreactor.

A composite gel system has been developed combining the chemical and physical properties of calcium alginate and agarose gels. The results of growing composite gel immobilized hybridoma SPO1 cells in a protein-free medium within a fluidized-bed perfusion bioreactor are presented in this paper. During the continuous operation of this system, the total cell density reached 3.9 x 10(7) cells per ml of beads (viability 79.6%). The specific productivity of monoclonal antibody of the immobilized hybridoma cells reached more than 1.5 micrograms per 10(6) viable cells per hour, compared with 0.5 for non-immobilized viable cells grown in a one liter agitated bioreactor with the same medium. Significant increases in cell metabolic activities, including substrate utilization and byproduct formation, were also observed. Leaching of materials from the beads was evident and the major fraction of released materials was alginate.

Simultaneous determination of ammonia nitrogen and L-glutamine in bioreactor media using flow injection.

A novel split stream flow injection (FI) system suitable for the simultaneous determination of L-glutamine and ammonia nitrogen (ammonia-N) in cell culture media is described. Potentiometric detection of ammonia-N in one portion of the manifold is achieved using a commercial ammonia gas-sensing electrode fitted with a wall-jet cap. L-Glutamine is quantified in the other part of the split sample by potentiometric detection of ammonium ions (by an ammonium-selective polymer membrane electrode), liberated from the hydrolysis of glutamine after the sample flows through a glass bead reactor containing immobilized glutaminase. Endogenous ammonia-N and potassium ions that would normally interfere with the glutamine measurement are removed upstream using a unique tubular cation-exchange unit. Using 50 microliters sample volumes and mixed solutions of ammonium chloride and L-glutamine in Iscove's Modified Dulbecco's Medium to calibrate the FI measuring system, values for ammonia-N and L-glutamine determined for 22 media samples obtained from a bioreactor growing retroviral producer cells correlate well with those measured with commercial, manual enzymic-spectrophotometric assay kits.

Mechanism of inhibition of cAMP-dependent epithelial chloride secretion by phorbol esters.

In T84 cells, we investigated how stimulation of protein kinase C leads to an inhibition of cAMP-dependent chloride secretion. Specifically, we tested the hypothesis that the inhibition was caused by loss of the cystic fibrosis transmembrane regulator (CFTR), an apical membrane chloride channel. As described by others (Trapnell, B. C., Zeitlin, P. L., Chu, C.-S., Yoshimura, K., Nakamura, H., Guggino, W. B., Bargon, J., Banks, T. C., Dalemans, W., Pavirani, A., Lecocq, J.-P., and Crystal, R. G. (1991) J. Biol. Chem. 266, 10319-10323), we found that treatment with the phorbol ester, phorbol myristate acetate (PMA), reduced CFTR mRNA levels by approximately 80% with a t 1/2 of approximately 2 h. Chloride secretion, measured as forskolin-induced short circuit current, was also abolished by PMA with a t 1/2 of approximately 2 h. Levels of mature glycosylated CFTR measured by Western blotting also declined to 50 +/- 8% (n = 7) of control after a 12-h PMA treatment. However, a 12-h exposure to PMA did not affect the forskolin-stimulated efflux of 125I into high potassium medium, a measure of apical membrane CFTR activity. We conclude that increased turnover of apical membrane CFTR in PMA-treated cells compensates for the decline in anion channel numbers. By contrast to its lack of effect on 125I effluxes, PMA reduced the cAMP-induced increase in 86Rb efflux, suggesting that it inhibits chloride secretion mainly by an action on basolateral potassium channels.

Rapid procedure for obtaining tracheal apical membranes.

Apical membranes from cow tracheal epithelium were prepared in a two-step process. First, most of the unwanted membranes in the crude homogenate were aggregated with Mg and removed by a low-speed spin. The membranes remaining in the supernatant were pelleted by a high-speed spin, resuspended, and exposed to ouabain-affinity chromatography. This step removed approximately 50% of the protein, all the Na-K-adenosinetriphosphatase, but had no effect on total levels of alkaline phosphatase (a marker for apical membranes). The specific activity of the apical membrane marker, alkaline phosphatase, was 21 +/- 7-fold (mean +/- SD) greater in the apical membranes than in the homogenate. Markers for nuclei, mitochondria, and basolateral membranes were excluded compared with the homogenate. Similar results were obtained with primary cultures of cow tracheal epithelium. The vesicular nature of the membranes was demonstrated in isotope uptake studies that revealed an osmotically active space.