Liver regeneration following repeated reversible portal vein embolization in an experimental model.

BACKGROUND: Portal vein embolization (PVE) is used routinely to prevent postoperative liver failure as a result of anticipated insufficient future liver remnant volume following resection. The authors have recently developed a technique for temporary PVE. The aim of this study was to assess the effect of repeated reversible PVE on hepatocyte proliferation and subsequent liver hypertrophy in rodents. METHODS: Four treatments were compared (n = 21 rats per group): single reversible PVE, two PVEs separated by 14 days, partial portal vein ligation or sham procedure. The feasibility and tolerance of the procedure were assessed. Volumetric imaging by CT was used to estimate the evolution of liver volumes. After death, the weight of liver lobes was measured and hepatocyte proliferation evaluated by immunostaining. RESULTS: Embolization of portal branches corresponding to 70 per cent of total portal flow was performed successfully in all animals. Repeated PVE induced additional hepatocyte proliferation. Repeated embolization resulted in superior hepatocyte proliferation in the non-occluded segments compared with portal vein ligation (31·1 versus 22·2 per cent; P = 0·003). The non-occluded to total liver volume ratio was higher in the repeated PVE group than in the single PVE and sham groups (P = 0·050 and P = 0·001 respectively). CONCLUSION: Repeated reversible PVE successfully induced additional hepatocyte proliferation and subsequent liver hypertrophy. Surgical relevance Portal vein embolization (PVE) is used routinely to prevent postoperative liver failure as a result of anticipated insufficient future liver remnant volume following resection. In the present study, a technique of repeated temporary PVE was developed in a rat model; this induced additional hepatocyte proliferation and an increase in liver volume compared with single embolization. This novel approach might help induce major hypertrophy of the future remnant liver, which could increase the rate of patients amenable to major liver resections.

Cholesterol synthesis-related enzyme oxidosqualene cyclase is required to maintain self-renewal in primary erythroid progenitors.

OBJECTIVES: Molecular mechanisms controlling cell fate decision making in self-renewing cells are poorly understood. A previous transcriptomic study, carried out in primary avian erythroid progenitor cells (T2ECs), revealed that the gene encoding oxidosqualene cyclase (OSC/LSS), an enzyme involved in cholesterol biosynthesis, is significantly up-regulated in self-renewing cells. The aim of the present work is to understand whether this up-regulation is required for self-renewal maintenance and what are the mechanisms involved. MATERIALS AND METHODS: To investigate OSC function, we studied effects of its enzymatic activity inhibition using Ro48-8071, a specific OSC inhibitor. In addition, we completed this pharmacological approach by RNAi-mediated OSC/LSS knockdown. The study of OSC inhibition was carried out on both self-renewing and differentiating cells to observe any state-dependent effect. RESULTS: Our data show that OSC acts both by protecting self-renewing T2EC cells from apoptosis and by blocking their differentiation program, as OSC inhibition is sufficient to trigger spontaneous commitment of self-renewing cells towards an early differentiation state. This is self-renewal specific, as OSC inhibition has no effect on erythroid progenitors that have already differentiated. CONCLUSIONS: Taken together, our results suggest that OSC/LSS expression and activity are required to maintain cell self-renewal and may be involved in the self-renewal versus differentiation/apoptosis decision making, by keeping cells in a self-renewal state.

Ischemic preconditioning induces autophagy and limits necrosis in human recipients of fatty liver grafts, decreasing the incidence of rejection episodes.

Whether ischemic preconditioning (IP) reduces ischemia/reperfusion (I/R) injury in human normal and fatty livers remains controversial. We compared two independent groups of liver donor transplants with versus without steatosis to evaluate IP consequences. Liver donors with (n=22) or without (n=28) steatosis either did or did not undergo IP before graft retrieval. Clinical data from the recipients, as well as histological and immunohistological characteristics of post-reperfusion biopsies were analyzed. Incidence of post-reperfusion necrosis was increased (10/10 versus 9/14, respectively; P<0.05) and the clinical outcome of recipients was worse for non-IP steatotic liver grafts compared with non-IP non-steatotic grafts. IP significantly lowered the transaminase values only in patients receiving a non-steatotic liver. An increased expression of beclin-1 and LC3, two pro-autophagic proteins, tended to decrease the incidence of necrosis (P=0.067) in IP steatotic livers compared with non-IP steatotic group. IP decreased the incidence of acute and chronic rejection episodes in steatotic livers (2/12 versus 6/10; P=0.07 and 2/12 versus 7/10; P<0.05, respectively), but not in non-steatotic livers. Thus, IP may induce autophagy in human steatotic liver grafts and reduce rejection in their recipients.

Basal endothelial nitric oxide synthase (eNOS) phosphorylation on Ser(1177) occurs in a stable microtubule- and tubulin acetylation-dependent manner.

To better understand the relationship between the subcellular compartmentalization of endothelial nitric oxide synthase (eNOS) and its function in endothelial cells, we addressed the roles of the microtubule network and of its dynamics in organizing Golgi-bound eNOS. We found that part of Golgi-bound eNOS localizes to the trans-Golgi network and/or to trans-Golgi network-derived vesicles and membrane tubules that are organized preferentially by stable microtubules. Also, while most of cellular eNOS was recovered in a detergent-resistant microtubule-enriched subcellular fraction, its recovery was impaired after total microtubule disassembly, but not after selective disassembly of dynamic microtubules or after microtubule stabilization. Basal eNOS phosphorylation on Ser(1177) further required the association of the trans-Golgi network to stable microtubules and was enhanced by microtubule stabilization. We finally show that the involvement of stable microtubules in basal eNOS phosphorylation involved alpha-tubulin acetylation. Microtubule-dependent organization of subcellular eNOS and control over its phosphorylation would thus be essential for endothelial cells to maintain their basal eNOS function.

[Clinical, haemodynamic and genetic features of familial pulmonary arterial hypertension]. / Caractéristiques cliniques, hémodynamiques et génétiques de l'hypertension artérielle pulmonaire familiale.

INTRODUCTION: Pulmonary arterial hypertension (PAH) is defined by a raised pressure in the pulmonary arterial circulation associated with small vessel narrowing due to proliferation of the endothelium and vascular smooth muscle. Idiopathic PAH should be distinguished from PAH associated with a causal disease. One familial type (familial PAH), gathered from one family, has recently been linked to a mutation of the BMPR 2 (bone morphogenetic protein receptor 2) gene. It seems important to compare the idiopathic form of PAH with these familial forms to confirm that the same diagnostic and therapeutic principles can be applied to familial PAH. MATERIAL AND METHODS: The demographic, clinical, haemodynamic and prognostic data from 34 cases of familial PAH were compared with those of 451 cases of idiopathic PAH. The genetic characteristics of the familial forms were also defined. RESULTS: Familial PAH presented at a younger age than idiopathic PH (31 +/- 15 vs. 45 +/- 18 years p=0.002) without any other demographic difference (sex-ratio 2.09/1 et 1.42/1 p=NS). There was no difference in exercises tolerance (6 minute walking test 341 +/- 98 and 289 +/- 135 metres p=NS), in haemodynamic parameters (mean PAP 65 +/- 12 and 62 +/- 15 mmHg, p=NS), or in prognosis, with the exception of an absence of a vasodilator response in the familial group to nitric oxide challenge. We found the BMPR 2 gene mutation to be quantitatively and qualitatively comparable to previously published data. CONCLUSION: The only difference between these two forms of this illness were of a younger age at presentation and an absent vasodilator response in the familial PAH group. We do not propose that familial PAH should be treated any differently from the idiopathic form. Genetic counselling will need to be developed in line with the progress being made in the understanding of this condition.

Allelic heterogeneity of glycogen storage disease type Ib in French patients: a study of 11 cases.

Eleven patients with glycogen storage disease type Ib (GSD Ib) were studied. Using a combination of single-strand conformation polymorphism (SSCP) analysis, restriction enzyme digestion and direct sequencing, we were able to identify 21/22 mutant alleles comprising 12 different mutations in the glucose-6-phosphate translocase gene (G6PT). Among these, one is a novel mutation of G6PT: 855T>C (L229P).

[Hormone determination in the management and monitoring of cycles of medically-assisted reproductive technology: value and difficulties of use]. / Les dosages hormonaux dans la prise en charge et le monitorage des cycles d'assistance médicale à la procréation: intérêt et difficultés de réalisation.

In assisted reproductive technology (ART), serum levels of several key hormones are used to evaluate ovarian follicular reserve and to monitor gonadotropin-stimulated follicle growth. Currently, serum estradiol, FSH, LH and inhibin B levels are examined and combined at the beginning of the menstrual cycle to evaluate the functional status of the ovaries, providing information for appropriate ovarian stimulation treatment and prognosis for in vitro fertilization (IVF) outcome. In women undergoing in vitro fertilization and embryo transfer (IVF-ET), ovulation stimulation is monitored by serial measurements of estradiol, progesterone and LH to monitor follicular growth, evaluate the progression of stimulation, adjust daily gonadotropin therapy for each patient, and predict the optimal day for the induction of ovulation. We analyze the importance of each hormone and discuss the discrepancies frequently reported resulting from differences in methods.

Impact of elements containing glycopeptide resistance genes on expression of virulence in Enterococcus faecalis peritonitis: a pilot study with rats.

The relationship between virulence and chromosomal elements containing glycopeptide resistance genes was experimentally assessed for two transconjugant strains of Enterococcus faecalis (VanA and VanB phenotypes) and compared to that for a susceptible wild-type strain. Microbiologic and inflammatory effects were assessed in a polymicrobial rat model of peritonitis. Mean peritoneal enterococcus concentrations +/- standard deviations at day 1 were 2.1 +/- 1.9, 1.3 +/- 1.1, and 1.7 +/- 2.0 log(10) CFU/ml for susceptible, VanA, and VanB strains, respectively (P < 0.05). At day 3 also there were lower concentrations of glycopeptide-resistant enterococcal strains in peritoneal fluid (3.2 +/- 3.4, 1.8 +/- 1.8, and 2.1 +/- 2.4 log(10) CFU/ml for susceptible, VanA, and VanB strains, respectively [P < 0.05]). Transconjugant glycopeptide-resistant strains were associated with increased peritoneal cell counts at the different evaluation times of the experiment (P < 0.001). Plasma alpha1-acid glycoprotein concentrations were lower in the presence of the susceptible strain (667 +/- 189 mg/liter) than in the presence of the VanA or VanB strain (1,193 +/- 419 or 1,210 +/- 404 mg/liter, respectively [P < 0.05]), while concentrations of tumor necrosis factor alpha and interleukin-6 in peritoneal fluid remained similar for the strains. These results suggest a trend toward variation of virulence of transconjugant strains compared to the wild-type strain in this peritonitis model.

The Golgi complex is a microtubule-organizing organelle.

We show that the Golgi complex can directly stimulate microtubule nucleation in vivo and in vitro and thus behaves as a potent microtubule-organizing organelle in interphase cells. With the use of nocodazole wash-out experiments in hepatic cells, we found that the occurrence of noncentrosomal, early stabilized microtubules is highly correlated with the subcellular localization of Golgi membranes. With the use of in vitro reconstituted microtubule assembly systems with or without cytosol, we also found that, in contrast to centrosomally attached microtubules, the distal ends of Golgi-attached microtubules are remotely stabilized in a way that requires additional cytosolic component(s). Finally, we demonstrate that Golgi-based microtubule nucleation is direct and involves a subset of gamma-tubulin bound to the cytoplasmic face of the organelle.

Polarized entry of uropathogenic Afa/Dr diffusely adhering Escherichia coli strain IH11128 into human epithelial cells: evidence for alpha5beta1 integrin recognition and subsequent internalization through a pathway involving caveolae and dynamic unstable microtubules.

Afa/Dr diffusely adhering Escherichia coli strain IH11128 bacteria basolaterally entered polarized epithelial cells by a CD55- and CD66e-independent mechanism through interaction with the alpha5beta1 integrin and a pathway involving caveolae and dynamic microtubules (MTs). IH11128 invasion within HeLa cells was dramatically decreased after the cells were treated with the cholesterol-extracting drug methyl-beta-cyclodextrin or the caveola-disrupting drug filipin. Disassembly of the dynamically unstable MT network by the compound 201-F resulted in a total abolition of IH11128 entry. In apically infected polarized fully differentiated Caco-2/TC7 cells, no IH11128 entry was observed. The entry of bacteria into apically IH11128-infected fully differentiated Caco-2/TC7 cells was greatly enhanced by treating cells with Ca2+-free medium supplemented with EGTA, a procedure that disrupts intercellular junctions and thus exposes the basolateral surface to bacteria. Basally infected fully differentiated polarized Caco-2/TC7 cells grown on inverted inserts mounted in chamber culture showed a highly significant level of intracellular IH11128 bacteria compared with cells subjected to the apical route of infection. No expression of CD55 and CD66e, the receptors for the Afa/Dr adhesins, was found at the basolateral domains of these cells. Consistent with the hypothesis that a cell-to-cell adhesion molecule acts as a receptor for polarized IH11128 entry, an antibody blockade using anti-alpha5beta1 integrin polyclonal antibody completely abolished bacterial entry. Experiments conducted with the laboratory strain E. coli K-12 EC901 carrying the recombinant plasmid pBJN406, which expresses Dr hemagglutinin, demonstrated that the dra operon is involved in polarized entry of IH11128 bacteria. Examined as a function of cell differentiation, the number of internalized bacteria decreased dramatically beyond cell confluency. Surviving intracellular IH11128 bacteria residing intracellularly had no effect on the functional differentiation of Caco-2/TC7 cells.

Hepatocyte differentiation of WIF-B cells includes a high capacity of interleukin-6-mediated induction of alpha 1-acid glycoprotein and alpha 2-macroglobulin.

Responsiveness to cytokine-mediated acute inflammatory stimuli of the highly differentiated and polarized WIF-B hybrid cell line was studied by measuring the induction of alpha 1-acid glycoprotein and alpha 2-macroglobulin mRNAs after interleukin-1, interleukin-6 and tumor necrosis factor-alpha treatments in the presence of dexamethasone. Compared with their Fao parent, WIF-B cells were 10 times more responsive to 24-h interleukin-6 induction regarding alpha 2-macroglobulin induction. At variance from the response measured in Fao cells, the late effects of interleukin-6 treatment confirmed the higher sensitivity of WIF-B cells to this cytokine as a 72-h treatment as 10 times more effective than a 24-h treatment at inducting alpha 1-acid glycoprotein mRNA. These findings highlight the hepatocyte differentiation of WIF-B cells compared with other hepatoma cell lines, with respect to the regulation of acute-phase protein gene expression. They also make WIF-B cells a convenient model to study the molecular effects of interleukin-6 in terms of transduction and/or transcription, and the many cross-talks that occur during the regulation of acute-phase protein gene expression.

Functional specialization of stable and dynamic microtubules in protein traffic in WIF-B cells.

We found that the magnesium salt of ilimaquinone, named 201-F, specifically disassembled dynamically unstable microtubules in fibroblasts and various epithelial cell lines. Unlike classical tubulin- interacting drugs such as nocodazole or colchicine which affect all classes of microtubules, 201-F did not depolymerize stable microtubules. In WIF-B-polarized hepatic cells, 201-F disrupted the Golgi complex and inhibited albumin and alpha1-antitrypsin secretion to the same extent as nocodazole. By contrast, 201-F did not impair the transport of membrane proteins to the basolateral surface, which was only affected by the total disassembly of cellular microtubules. Transcytosis of two apical membrane proteins-the alkaline phosphodiesterase B10 and dipeptidyl peptidase IV-was affected to the same extent by 201-F and nocodazole. Taken together, these results indicate that only dynamically unstable microtubules are involved in the transport of secretory proteins to the plasma membrane, and in the transcytosis of membrane proteins to the apical surface. By contrast, stable microtubules, which are not functionally affected by 201-F treatment, are involved in the transport of membrane proteins to the basolateral surface. By specifically disassembling highly dynamic microtubules, 201-F is an invaluable tool with which to study the functional specialization of stable and dynamic microtubules in living cells.

Inducible expression of the alpha1-acid glycoprotein by rat and human type II alveolar epithelial cells.

Alpha1-acid glycoprotein (AGP) is a major acute phase protein in rat and human. AGP has important immunomodulatory functions that are potentially important for pulmonary inflammatory response. The liver is the main tissue for AGP synthesis in the organism, but the expression of AGP in the rat lung has not been investigated. We show that AGP mRNA was induced in the lung of dexamethasone-, turpentine-, or LPS-treated rats, whereas AGP mRNA was not detected in the lung of control rats. In the lung of animals treated intratracheally with LPS, in situ hybridization showed that AGP gene expression was restricted to cells located in the corners of the alveolus, consistent with an alveolar type II (ATII) cell localization. The inducible expression of the AGP gene was confirmed in vitro with SV40 T2 cells and rat ATII cells in primary culture: maximal expression required the presence of dexamethasone. IL-1 and the conditioned medium of alveolar macrophages acted synergistically with dexamethasone. Rat ATII cells secreted immunoreactive AGP in vitro when stimulated with dexamethasone or with a combination of dexamethasone and the conditioned medium of alveolar macrophages. In vivo, in the human lung, we detected immunoreactive AGP in hyperplastic ATII cells, whereas we did not detect AGP in the normal lung. We conclude that AGP is expressed in the lung in cases of inflammation and that ATII cells are the main source of AGP in the lung.

Isoenzymatic diagnosis of filariae: a method for separation of lactate dehydrogenase isoenzymes from Molinema dessetae (Nematoda: Filarioidea).

Lactate dehydrogenase (LDH) is highly active in filariae and could be a valuable tool for phyllogeny studies. Unfortunately, the isoenzymatic diagnosis of filariae is often difficult for LDH because of a poor mobility of the enzymes in starch gels which are the most commonly used in such studies. We propose here a method to separate filarial LDH isoenzymes using disc electrophoresis. The experiments were carried out on male and female Molinema dessetae in order to compare their respective isoenzymes. The study of several parameters such as buffer systems, percentage of bisacrylamide and progression time led to optimize the enzyme separation. LDH from male and female filariae were compared to mammal LDH-H4 and LDH-M4. Five and four LDH isoenzymes were found, respectively, in male and female worms. Relative concentration of each isoenzyme diverged between male and female worms. Mammal muscle LDH-M4 type moved between LDH2 and LDH3 from female worms, and between LDH1 and LDH2 from male worms. Mammal heart H4 type enzyme was very different in electrophoretic mobility. The ratio of each isoenzyme was determined by densitometry. The major isoenzymes from female worms will be studied as a biochemical target for chemotherapeutic attack.

Differential secretion of alpha 1-acid glycoprotein occurs in the Golgi complex of isolated rat hepatocytes. Evidence of partial retention in the Golgi.

Using weakly basic amines, we investigated the step at which the secretion kinetics of concanavalin-A-retained and nonretained alpha 1-acid glycoprotein glycoforms diverge in isolated rat hepatocytes. Both chloroquine and primaquine, whose action on protein secretion is targeted to terminal domains of the Golgi apparatus, cancelled the kinetic difference without influencing carbohydrate chain sialylation. To test for a possible interaction of alpha 1-acid glycoprotein with Golgi membranes, we also permeabilized control and primaquine-treated hepatocytes, as well as purified Golgi preparations, with saponin. In each case, we found that alpha 1-acid glycoprotein was associated with Golgi membranes, the association being more marked in primaquine-treated cells than in control cells. Membrane-bound alpha 1-acid glycoprotein appeared to be preferentially retained on concanavalin A. Such retention could account for the divergent secretion kinetics of alpha 1-acid glycoprotein glycoforms.

Elution mode of Pneumocystis carinii cysts in gravitational field-flow fractionation.

The simplest field-flow fractionation technique, i.e. gravitational, was used in an attempt to purify a Pneumocystis carinii cyst suspension. This parasite is an opportunistic invader in immunocompromised patients, especially those suffering from AIDS. The cyst stage is spherical and 5 microns in diameter. Unexpected retention times, not systematically related to the size and the density of the parasite, were obtained under various experimental conditions. When silicone-coated walls were used, Pneumocystis carinii cysts were eluted in the void volume, whereas when uncoated walls were used with a sodium dodecyl sulphate-enriched carrier phase, retention was observed. These phenomena are probably related to the high degree of hydrophobicity of these micrometre-sized biological particles; this degree can be easily determined. The use of the gravitational field-flow fractionation technique can be of a great interest for the development of new methods for diagnostic purposes. Particle-wall interactions and their modifications due to the carrier phase or to the wall treatment can be employed in the search for new bronchoalveolar lavage solutions.

Recombinant human interleukin 1 beta and tumor necrosis factor affect glycosylation of serum alpha 1-acid glycoprotein in rats.

Serum concentration and glycosylation of rat alpha 1-acid glycoprotein (alpha 1-AGP) were evaluated after the in vivo administration of recombinant human interleukin-1 beta (rhIL-1 beta) and tumor necrosis factor alpha (rhTNF-alpha), alone or associated. The effect of LPS and turpentine was also studied. In all models, serum alpha 1-AGP concentrations were increased and glycosylation was altered. The alpha 1-AGP levels reached 1.8 g/liter with cytokines alone, 2.1 g/liter with cytokines associated or LPS, and 3.4 g/liter with turpentine. Analysis by concanavalin A (Con A) affinoimmunoelectrophoresis (CAIE) revealed that the relative proportion of Con A unreactive form always decreased whatever the inducing agent. On the other hand, the resulting effect on the concentrations of Con A unreactive alpha 1-AGP concentrations was an increase with cytokines alone or LPS and a decrease with cytokines associated or turpentine. These results suggest a dissociation between the alteration in the level of alpha 1-AGP synthesis and in the pattern of its glycosylation in the various inflammatory models.

Differential rates of glycoprotein secretion by isolated rat hepatocytes studied in terms of concanavalin A binding.

Using a concanavalin-A-based method which respects cell function, we have shown that the kinetics of glycoprotein secretion appear to depend on the nature of the oligosaccharide moiety. In 37 degrees C pulse/chase experiments using freshly isolated normal rat hepatocytes, we found that except for transferrin, whose rate of secretion was independent of its concanavalin A reactivity, the secretion of the concanavalin-A-retained forms of alpha 1 acid glycoprotein, T-kininogen, alpha 1 protease inhibitor and alpha 1 inhibitor III was slower than that of the concanavalin-A-non-retained forms. When hepatocytes were incubated at 20 degrees C, secretion was blocked with the accumulation of mainly endoglycosidase-H-sensitive forms. The secretion kinetics of the concanavalin-A-differentiated forms were still different when the temperature was shifted back to 37 degrees C. The divergence between the secretion rates of the concanavalin-A-differentiated forms would appear to be due to a late event in intracellular protein trafficking, which may depend on the sugar content and/or the number of carbohydrate chains of the glycoproteins.

Modification of inflammatory processes by phenobarbital in rats.

Enzyme-inducing drugs such as phenobarbital (PB) increase serum concentrations of an acute-phase protein, alpha 1-acid glycoprotein (AGP), in man, dogs, and rats via an unknown mechanism. We studied the effects of PB on components of an acute inflammatory reaction in rats in order to determine if PB acts only on this biological marker of inflammation or is capable of altering the clinical course of inflammatory processes. Local carrageenan injection induces a similar time-dependent plantar edema and increases serum AGP levels in Sprague-Dawley (SD) and Dark Agouti (DA) rats. Pretreatment with PB for seven days modified neither parameter in SD rats while plantar edema was aggravated and serum AGP levels were increased in DA rats. The sedative-hypnotic properties of PB were not involved, since a single administration of this drug had no action in DA rats. On the other hand, chronic PB administration reduced the severity of an autoimmune disease, type II collagen-induced arthritis, in DA rats. These data indicate that PB, a potent inducer a cytochrome P-450-dependent enzymes, modifies the course of the inflammatory process. Preliminary results with macrophage transfer experiments suggest that this response to PB could be mediated by stimulated macrophages.