Urinary F2-isoprostanes formation in kidney transplantation.

BACKGROUND: Oxygen free-radical mediated lipid peroxidation has been implicated in many diseases such as chronic renal failure, hemodialysis and chronic kidney transplant rejection. However, insight into the role of free radical generation in kidney transplantation has been constrained by the limitations of current indexes of oxidant stress in vivo. Isoprostaglandin F2alpha type-III (iPF2alpha-III, formerly known as 8-iso-prostaglandin F2alpha) is emerging as a reliable marker of oxidant stress in vivo. The purpose of our study was to investigate iPF2alpha-III formation as an index of lipid peroxidation in the 5 d following kidney transplantation. METHODS: Urinary iPF2alpha-III measurements were performed by enzyme immunoassay from day I to 5 in 11 patients undergoing kidney transplantation. Results were compared with 11 healthy volunteers matched in sex, age and cigarette smoking. RESULTS: Urinary excretion of iPF2alpha-III at day 1 did not significantly differ between control and transplant group (111 +/- 17 vs. 92 +/- 10 pM/ mM creatinine, respectively, NS). Urinary iPF2alpha-III levels did not differ between day 1 to 5, and were not correlated to cold ischaemia time. CONCLUSION: Our study shows no evidence of enhanced lipid peroxidation in the first 5 d following kidney transplantation.

Combination treatment for chronic hepatitis C: what is the role of ribavirin?

Ribavirin in combination with interferon-alpha2b is the new standard for chronic hepatitis C (CHC) treatment. Although usually considered as an antiviral compound, this guanosine analogue shows some additional effects on the immune system that could largely contribute to its clinical efficacy in CHC. Numerous in vitro experiments demonstrate that ribavirin has a selective down-regulatory effect on TH2 cytokine release with, in some cases, a concomitant TH1 cytokine up-regulation. In vivo, combination treatment of CHC patients was shown to induce a predominant TH1 response in isolated PBMCs, but also a reduction of peripheral TH2 response. Considering that: 1) a strong CD(4)+ helper T-cell response is associated with viral clearance in acutely infected patients; 2) a weak T-cell response to the viral antigens is common in chronic infected patients; 3) responding patients to combination treatment (but not non-responding patients) altered their cytokine profile under treatment, either to express IFN-gamma or to reduce pro-inflammatory mediators; it is highly presumed that ribavirine participates to restore an efficient T-cell response and to reduce the non-specific inflammatory cytolytic activity during CHC combination treatment.

[Gene therapy approaches in chronic viral hepatitis]. / Approches de thérapie génique dans les hépatites virales chroniques.

The clinical and socioeconomic background of chronic viral hepatitis is favourable to new therapeutic approaches based on gene biochemistry. As with all gene therapy, the treatment of chronic viral hepatitis using this approach would make use of a therapeutic gene and a delivery system adapted to the pharmaceutical objectives of targeting, gene expression and kinetics. The various vectors under review are not yet sufficiently optimized for selective targeting of infected hepatocytes. Moreover, four therapeutic gene processes are currently under development: antisense oligonucleotides, ribozymes, dominant negative mutants and DNA vaccines. These developments are obviously limited by the lack of experimental or animal models representative of the pathophysiology of chronic viral hepatitis. The gene therapy for chronic viral hepatitis is nearly ready for clinical evaluation but must be weighed against the continuous progress of pharmaceutical treatments.

Human internal mammary artery contraction by isoprostaglandin f(2alpha) type-III [8-iso-prostaglandin F(2alpha)].

Isoprostaglandin F(2alpha) type-III (formerly known as 8-iso-prostaglandin F(2alpha)) is produced in large quantities in vivo in clinical situations associated with oxidant stress such as atherosclerosis, hypercholesterolemia, and myocardial reperfusion. Isoprostaglandin F(2alpha) type-III may alter smooth muscle and platelet functions. The aim of this study was to evaluate the effects of isoprostaglandin F(2alpha) type-III on isolated human internal mammary arteries, and to characterise the signalling underlying mechanisms. In organ baths, concentration-dependent contractions of human internal mammary arteries were obtained in response to isoprostaglandin F(2alpha) type-III stimulation. The responses to isoprostaglandin F(2alpha) type-III were inhibited in a concentration-dependent manner by the thromboxane A(2) receptor antagonist, GR 32191 ([1R-[1 alpha(Z), 2beta,3beta,5 alpha(+)-7-[[1, 1'-biphenyl)-4-yl]methoxy]-3-hydroxy-2-(1-piperidinyl) cyclo pentyl]-4-4heptanoic acid], hydrochloride), 3x10(-9) to 3x10(-7) M). However, this effect was associated with a decreased maximal contraction. AH 6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid, 10(-6) to 3x10(-5) M), an EP(1)-DP receptor antagonist had no effect on isoprostaglandin F(2alpha) type-III-induced contractions. The maximal responses to isoprostaglandin F(2alpha) type-III were significantly reduced in the presence of the cyclooxygenase inhibitor indomethacin (10(-5) M) (E(max): 147+/-20% vs. 213+/-19% in control group, P<0.05). Isoprostaglandin F(2alpha) type-III stimulated thromboxane B(2) release (5.7-fold increase) from human internal mammary arteries. Baicaleine, a non-specific lipoxygenase inhibitor, (10(-4) M) and AA 861 (2,3,5-trimethyl-6-(12-hydroxy-5, 10-dodecadiynyl)-1,4 benzoquinone), a 5-lipoxygenase inhibitor (10(-5) M) did not affect isoprostaglandin F(2alpha) type-III response. In conclusion, this study shows that (1) isoprostaglandin F(2alpha) type-III is a vasoconstrictor in human internal mammary arteries, with a potency equivalent to prostaglandin F(2alpha), (2) the contractions induced by isoprostaglandin F(2alpha) type-III are mediated by TP receptor but not EP(1)-DP-receptor activation, (3) thromboxane A(2) but not cysteinyl leukotrienes production is involved in the vascular effects of isoprostaglandin F(2alpha) type-III. Isoprostaglandin F(2alpha) type-III, produced at sites of free radical generation, may play an important role in internal mammary artery spasm in situations of oxidant stress such as coronary bypass surgery.

[Isoprostanes: new markers of oxidative stress in human diseases]. / Isoprostanes: nouveaux marqueurs du stress oxydant en pathologie humaine.

BACKGROUND: Most of the traditional methods used to assess oxidative stress in clinical setting are non specific, unreliable or inaccurate. Recently, a novel family of prostaglandin F2 isomers, called F2-isoprostanes, produced in vivo by a free radical peroxidation of arachidonic acid, has been described. These compounds may produce physiological or pathological effects due to their ability to alter smooth muscle and platelet functions. The quantification of the two major isoforms (isoprostaglandin F2 alpha type-III and VI) in biological fluids and tissues as markers of lipid peroxidation appears to be an important advance in our ability to explore the role of free radicals in the pathogenesis of human disease. CLINICAL DATA: Urinary excretion of F2-isoprostanes is correlated with age, indicating increased oxidative stress during the normal aging process. High F2-isoprostanes concentration has been described in diseases such as ischemic heart disease, diabetes, Alzheimer's disease and hepatic cirrhosis. The correlation of F2-isoprostane concentrations and human diseases severity in hepatic cirrhosis, cardiac failure and diabetes suggest that these compounds may be of interest as predictive markers. PERSPECTIVES: Preliminary studies suggest the use of F2-isoprostanes as prognosis markers. In addition, F2-isoprostanes quantification offers promising potential as intermediate endpoints for clinical studies of antioxidant therapies.

Are isoprostanes a clinical marker for antioxidant drug investigation?

Numerous pathological conditions are suspected to involve free radical production as part of their pathogenic process. Therefore, a pharmacological control of oxidative stress could probably benefit many vascular, inflammatory or degenerative diseases. However, the development of antioxidant drugs and their clinical evaluation are limited by the absence of an accurate, reliable and easy-to-handle marker of tissue oxidative events. Isoprostanes (isoPs), a prostaglandin-related series of metabolites, are emerging as major candidates for clinical measurement of oxidative stress. They are chemically stable products of lipid peroxidation, formed in cellular membranes and subsequently released and excreted in the urine. Many recent clinical studies have reported that urinary and plasma levels of isoPs (in particular the iPF2alpha-III isomer also called 8-epi-PGF2alpha) are increased in clinical conditions where oxidative stress is suspected to play a pathogenic role. Moreover, isoPs have been detected in tissue extracts from atherosclerotic plaques and Alzheimer patients brain tissue. Finally, antioxidant treatments such as vitamin E supplementation appear to reduce isoPs levels in biological fluids of treated patients. These preliminary observations argue for a further investigation of isoPs as a practical pharmacodynamic endpoint for the clinical evaluation of antioxidant therapies.

Protein tyrosine kinase activity as a prognostic parameter in breast cancer, a pilot study.

Protein tyrosine kinase (PTK) activity was assayed in cytosolic extracts from normal breast tissue, benign tumors, and 84 T1-T2, N0-N1 M0, breast carcinomas. Normal breast tissue extracts yielded an average value of 1.9 +/- 1.1 pmol 32P incorporated/min/mg protein, whereas a mean of 12.5 +/- 6.1 was obtained for cancer samples. With a median follow-up of 34 months, in the series of 40 patients classified N-, PTK positive patients presented a significantly smaller 3-year disease free survival than the PTK negative ones. Multivariate analysis shows that PTK activity emerges as a potential prognostic factor in breast cancer (p = 0.02). These preliminary results will be updated on a bigger cohort of patients.

Activation of protein kinase C in lipid monolayers.

The potential of lipid monolayers spread at an air-water interface was investigated as a well defined membrane model able to support protein kinase C (PKC) association and activation. PKC association to a mixed phospholipid film (phosphatidylcholine, phosphatidylserine) could be detected by an increase of the monolayer surface pressure. This association was strikingly dependent upon the presence of submicromolar concentrations of Ca2+. The effect of Ca2+ resulted in an increase of the PKC penetration into the lipid core at a given permissive surface pressure as well as in a marked increase of the critical surface pressure (29-38 dynes/cm) above which the enzyme was excluded from the membrane. Inclusion of diacylglycerol or tetradecanoate phorbol acetate (TPA) did not modify the PKC-monolayer association in a detectable manner. PKC associated to the lipid layer exhibited the expected catalytic property and was fully activated when diacylglycerol or TPA was included in the membrane. PKC activity was highly dependent upon the surface pressure of the lipid monolayer, being optimal between 30 and 35 dynes/cm. Study of the compression isotherm of various diacylglycerol structures revealed that all potent PKC agonists exhibited an expanded liquid phase behavior with collapse pressure below 40 dynes/cm, in contrast to weak activators which showed condensed isotherms with high collapse pressure (approximately equal to 60 dynes/cm). These observations showed that the lipid monolayer system is well adapted to the study of the molecular mechanisms involved in the regulation of PKC activity at a model membrane interface. They are in line with the suggestion of a major role of Ca2+ in the association (translocation) of PKC to membrane in living cell and suggest that diacylglycerol (and TPA) might activate membrane-associated PKC through local change in the surrounding lipid phase organization.

Estimation of epidermal growth factor receptor in 177 breast cancers: correlation with prognostic factors.

Steroid receptors (ER, PR) and EGF-receptor were determined on 177 loco-regional primary breast cancers. Binding assay for EGF receptor was performed using a single saturating concentration of 125I-EGF incubated with membranes in the presence or absence of unlabeled EGF. With threshold values of 5 fmol/mg and 10 fmol/mg for EGF-R and steroid receptors respectively, we noted an inverse relationship between the expression of EGF-R, and ER and PR. EGF-R expression is decreased with tumor differentiation.

Saliva from cystic fibrosis patients contains an unusual form of epidermal growth factor.

Epidermal Growth Factor (EGF) was assayed in saliva collected from control subjects and cystic fibrosis (CF) patients, using both radioimmuno (RIA) and radioreceptor (RRA) assays. An intriguing finding was that the average ratio of the values found by RRA over those obtained by RIA was of 1.7 for normal subjects and of about 0.4 for CF patients. This observation could be understood following gel filtration analysis of EGF-like material in these salivary fluids. Whereas control saliva contained the expected 6 kDa EGF active peptide, the immunoreactive EGF material from CF patients eluted as a polydisperse macromolecular moiety. The poor biological reactivity of this material as assessed by radioreceptor assay suggests that this EGF anomaly may contribute to the physiopathology of cystic fibrosis, especially as the upper gastrointestinal tract differentiated functions may be the target of normal salivary EGF.

Differential inhibition of protein kinase C subtypes.

Catalytic properties of protein kinase C isoforms purified from rat brain and bovine adrenocortical tissues were examined. The results showed that known inhibitors of PKC activity such as gossypol and H-7 were active on all the three isolated enzyme isoforms with similar IC50 values. However, whereas the type III brain isozyme activity was not affected by a preincubation with phosphatidylserine (PS), the same treatment resulted in a virtually complete loss of the type I and II isoform activities within 4 min at 30 degrees. This kinase inactivation caused by PS preincubation was prevented in the presence of ATP-Mg2+ or its competitive inhibitor H-7. These findings indicate that the type III isoform can clearly be distinguished from the other members of the PKC family by this specific property. This approach was used to confirm the characterization of the single form of PKC detected in bovine adrenocortical tissue as a type III isotype. This specific behavior toward phosphatidylserine suggests that the molecular organization of the phospholipid sensitive, regulatory domain of the PKC isoform III with regard to its catalytic site and thus its mechanism of activation may differ from that of other PKC isotypes.

Phospholipid independent phosphorylation of protamine by protein kinase C: effects of polyanions.

The ability of purified protein kinase C (PKC) to phosphorylate protamine sulphate was found to be totally independent of phospholipid cofactors, whereas the phosphorylation of protamine free base was markedly increased by the presence of phosphatidylserine (PS). The hypothesis of an activation of PKC by the sulphate groups of protamine sulphate was confirmed by the high phosphorylation of protamine free base in the presence of non-peptide polyanionic compounds, such as glycoaminoglycans or polynucleotides. The catalytic fragment of PKC supported protamine base phosphorylation with the same polyanionic dependency. Light scattering intensity measurements showed that this phosphorylation correlated to the substrate/cofactor aggregation. These data support the view that apparent phospholipid-independent activation of PKC results from the formation of aggregates in the assay and this could result in the non-specific activation of this enzyme through its catalytic domain.

Phorbol ester-induced alteration of protein kinase C catalytic properties occurs at the membrane level and is not reproduced by physiological stimuli.

Potent tumor promoter TPA (1-100 nM) has previously been shown to induce a striking alteration of protein kinase C catalytic properties in target cells (C. Cochet et al., 1986, Biochem. Biophys. Res. Comm. 134, 1031-1037). This alteration contributes to the apparent loss of cellular protein kinase C, secondary to TPA treatment, when the enzyme is probed by its phospholipid-dependent histone kinase activity. This effect was observed as well when rat-1 cells were treated by other tumor promoters such as mezerein, teleocidin, aplysiatoxin and palytoxin, whereas inactive phorbol ester structures were ineffective. On the other hand, 1,2-dioctanoyl glycerol did not induce that effect. This protein kinase C alteration was shown to occur at the cellular membrane level. It is suggested that membrane translocation and activation of protein kinase C induced by potent tumor promoter structures are not functionally equivalent to that secondary to physiological stimuli. Although the mechanisms underlying this phenomenon remains to be understood at the molecular level, it may be of significance in the process of tumor promotion.

Altered catalytic properties of protein kinase C in phorbol ester treated cells.

Exposure of various cell types (rat-1 fibroblasts, bovine adrenocortical cells, human lymphoid cells) to nanomolar concentrations of TPA, resulted in a rapid, apparent loss of cellular protein kinase C content, when the enzyme was assayed by its phospholipid and Ca2+-dependent histone (H1)-kinase activity, following solubilization and DEAE-cellulose chromatography isolation. By contrast, no loss of protein kinase C was detected when the enzyme was probed by its high affinity PDBu binding capacity nor when the kinase activity was assayed with protein substrates other than histones, such as vinculin and a cytochrome P-450. It is concluded that, in addition to the previously reported enzyme subcellular redistribution, following TPA treatment, the phorbol ester induces striking alterations of the cellular protein kinase C catalytic activities. The molecular mechanisms of these changes and their implication in the tumor promotion process remain to be clarified.