Trophic transfer of copper decreases the condition index in Crassostrea gigas spat in concomitance with a change in the microalgal fatty acid profile and enhanced oyster energy demand.

Due to new usages and sources, copper (Cu) concentrations are increasing in the Arcachon Basin, an important shellfish production area in France. In the present paper, the trophic transfer of Cu was studied between a microalga, Tetraselmis suecica, and Crassostrea gigas (Pacific oyster) spat. An experimental approach was developed to assess Cu exposure, transfer and toxicity on both phytoplankton and spat. Exposure of microalgal cultures to Cu for 7-8 days (3.1 ± 0.1, 15.7 ± 0.2 and 50.4 ± 1.0 µg Cu·L-1 for the control, Cu15 and Cu50 conditions, respectively) led to concentrations in microalgae (28.3 ± 0.9 and 110.7 ± 11.9 mg Cu·kg dry weight-1 for Cu15 and Cu50, respectively) close to those measured in the field. Despite Cu accumulation, the physiology of the microalgae remained poorly affected. Exposed cultures could only be discriminated from controls by a higher relative content in intracellular reactive oxygen species, and a lower relative content in lipids together with a reduced metabolic activity. By contrast, the fatty acid profile of microalgae was modified, with a particularly relevant lower content of the essential polyunsaturated fatty acid 22:6n-3 (docosahexaenoic acid [DHA]). Following 21 days of spat feeding with Cu15 and Cu50 microalgal cultures, trophic transfer of Cu was observed with a high initial Cu concentration in spat tissues. No effect was observed on oxidative stress endpoints. Cu exposure was responsible for a decrease in the spat condition index, an outcome that could be related to an insufficient DHA supply and extra energy demand as suggested by the overexpression of genes involved in energy metabolism, ATP synthesis and glycogen catabolism.

Effect of plasma fractions from patients with focal and segmental glomerulosclerosis on rat proteinuria.

BACKGROUND: Patients suffering from focal and segmental glomerulosclerosis (FSGS) and in whom this disease recurs after transplantation are likely to have an active form of the disease and to have a factor(s) (such as, albuminuric factor) present in their blood that alters glomerular permeability for albumin. METHODS: We used a sequential 50 and 70% ammonium sulfate (AS) precipitation of plasma from patients with relapsing FSGS and non-FSGS nephrotic syndrome (NS), in addition to plasma from healthy individuals, to obtain both an immunoglobulin (Ig)-rich fraction (50% AS precipitate) and a non-Ig fraction (70% AS supernatant). These fractions were injected intra-arterially or intravenously/intraperitoneally into Sprague-Dawley rats, and proteinuria (g protein/mmol creatinine) was measured for 24 hours. Ig fractions eluted from immunoadsorption onto protein A were also tested. A biochemical characterization was then carried out on the 70% AS supernatants by ultrafiltration on 30 and 50 kD cut-off membranes and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Differentially stained bands were sequenced. RESULTS: The 70% AS supernatants from FSGS patients induced proteinuria when injected intra-arterially into normal rats. This effect was significantly different (P < 0.05) from that observed when similar fractions were prepared from the plasma of patients suffering from non-FSGS NS, but was not different from that observed with fractions from healthy individuals and even with an injection of saline solution. Injections of other plasma fractions did not induce a significant proteinuria in the FSGS group versus the non-FSGS NS group. SDS-PAGE of 70% AS supernatants revealed a protein of 23 kD that was more concentrated in AS supernatants from FSGS plasma than the other plasma samples and that was identified by microsequencing as apolipoprotein A1. After sequential ultrafiltration of 70% AS supernatants on 30 and 50 kD cut-off membranes, a second band of 43 kD was found at a much higher concentration in the FSGS samples than in non-FSGS NS and healthy individuals samples. This band is likely to correspond to a candidate albuminuric factor recently reported by another group [1], and was identified by microsequencing as alpha1 acid glycoprotein or orosomucoid. Consequently, purified orosomucoid from the plasma of FSGS, non-FSGS NS patients, or healthy individuals was injected intra-arterially into rats. No differences were found between the proteinuria induced in each group. CONCLUSIONS: These data strongly suggest that in vivo injection of material into the rat is not a reliable model for testing plasma fraction activity and that the 43 kD orosomucoid is not likely to be the albuminuric factor.

Interaction of anti-HLA antibodies with pig xenoantigens.

BACKGROUND: Many patients with renal failure are condemned to long-term dialysis with little prospect of transplantation because they are highly sensitized with immunoglobulin G (IgG) directed against class I human leukocyte antigens (HLA) of virtually all donors. Xenotransplantation could represent an attractive solution providing their alloantibodies (alloAb) do not recognize porcine motifs. Hitherto there has been no in vivo demonstration of any cross-reactivity and the objective of this work was to investigate this problem using a technique of extracorporeal pig kidney perfusion as a model of clinical xenografting. METHODS: Pig kidneys were perfused ex vivo with plasma from both a group of highly sensitized patients and healthy individuals. Sequential plasma samples were analyzed for the titer of anti-Galalpha1-3Gal antibody (Ab) (major natural xenoreactive Ab) by enzyme-linked immunosorbent assay and anti-HLA class I Ab against a cell panel. At the end of perfusion, kidneys were perfused with a citric acid buffer to elute bound Ab. RESULTS: Galalpha1-3Gal Ab were shown to decrease rapidly in the plasma (in less than 10 min) and then reached a plateau. A fractional decrease in anti-HLA Ab was also found in some of the perfused plasma samples. Anti-Gal Ab were readily detected in all citric acid perfusates and anti-HLA Ab in 8 of 10. The HLA specificities of eluted Ab were mainly concordant with the originally designated specificities for each patient. CONCLUSION: Anti-HLA class I Ab presumably cross-react with pig class I homologues. However, some plasma samples did not cross-react, suggesting that negatively cross-matched pig kidneys could be identified in the pig population for xenotransplantation in these patients. Further studies are required to precisely describe these cross-reactivities and to understand their functional significance in xenotransplantation.

Antihuman immunoglobulin affinity immunoadsorption strongly decreases proteinuria in patients with relapsing nephrotic syndrome.

Approximately 20 to 30% of patients with idiopathic nephrotic syndrome and focal glomerulosclerosis experience a relapse of their nephrotic syndrome after transplantation. Previously, it has been shown that ex vivo immunoadsorption on protein A strongly (although transiently) reduces proteinuria in relapsing patients. To investigate whether the factor(s) that give rise to albuminuria are bound directly to protein A in the immunoadsorption procedure or are part of a complex with Ig, four patients with relapse of focal glomerulosclerosis presenting as nephrotic syndrome after transplantation were treated, sequentially, using a (non-protein A) anti-Ig affinity column and a protein A column. This study reports that the effect on proteinuria of immunoadsorption using an anti-Ig immunoaffinity column is comparable in its magnitude and kinetics to that of immunoadsorption on protein A. The two procedures were also equally effective in depleting the relapsing patients' plasma of a factor capable of altering the albumin permselectivity of isolated glomeruli in vitro. This study demonstrates for the first time that immunoglobulins have a role in the nephrotic syndrome. In addition, the fact that the two different immunoadsorption procedures both resulted in the removal of the same putative albuminuric factor in these patients and that no autoreactivity of eluted immunoglobulins was observed on human tissues strongly suggests that the factor or factors that may be responsible for immediate nephrotic syndrome after transplantation are bound to an immunoglobulin. However, no firm evidence can be yet provided against a direct role of immunoglobulins.

Plasma immunadsorption treatment in patients with primary focal and segmental glomerulosclerosis.

BACKGROUND: In primary focal and segmental glomerulosclerosis (FSGS) renal prognosis is poor if no remission of proteinuria can be obtained by treatment. In some patients a permeability factor, responsible for damaging the glomerular epithelial cell and detectable by an in vitro test (GVV-test), seems to be present in the serum. METHOD: We determined the effects of an immunadsorption treatment (IAT) on proteinuria and glomerular permselectivity (using a neutral dextran and dextransulfate-sieving technique to assess glomerular size and charge selectivity) in five patients with FSGS in the native kidneys and three patients with recurrence of FSGS after kidney transplantation. Furthermore, we performed the GVV-test using sera obtained from the patients before and after therapy. RESULTS: IAT reduced proteinuria by more than 50% in four patients, all of whom had an improvement in glomerular-size selectivity. Charge selectivity was better preserved after therapy in three out of these four subjects. The GVV-test prior to IAT was positive in two patients who also responded clinically to therapy. After IAT the GVV-test was negative in all patients, indicating an elimination of the proteinuric factor in the two previously positive patients. CONCLUSION: We conclude that a positive GVV-test before treatment makes a favourable response of IAT on proteinuria likely in patients with FSGS. If a reduction of proteinuria can be obtained by IAT this is due to an improvement in glomerular size and/or charge selectivity.

Study of the in vitro effect on glomerular albumin permselectivity of serum before and after renal transplantation in focal segmental glomerulosclerosis.

BACKGROUND: The selective proteinuria observed in patients with focal segmental glomerulosclerosis (FSGS) suggests an abnormal loss of fixed anionic charges on the glomerular capillary wall. METHODS: In this article, we have studied the putative presence of such factor(s) by using a new in vitro assay to assess glomerular permselectivity by measuring glomerular volume variation (GVV) in isolated glomeruli after hypotonic stress. We randomly tested the serum GVV activity of 10 healthy donors and 143 patients before transplantation. Of the patients, 80 had FSGS, 26 membranous glomerulonephritis, 19 polycystic kidney disease, and 18 malformative uropathies. Moreover, we tested the pre- and posttransplantation serum of 14 patients with recurrence and 14 without recurrence. RESULTS: Serum GVV was significantly higher in patients with FSGS than in those with the other end-stage renal diseases studied (P<0.01) or in healthy donors (P<0.01). However, a wide distribution of serum GVV activity in patients with and without FSGS was observed. Statistically, pregraft GVV values were not predictive of the recurrence of FSGS after transplantation. Moreover, we observed a significant decrease in serum GVV activity after transplantation in patients without recurrence (P<0.01) compared to those who underwent a recurrence. CONCLUSIONS: These results reinforce the hypothesis of a circulating factor that alters glomerular albumin permselectivity in FSGS patients. However, the presence of this factor before transplantation did not appear to predict relapse of the disease after transplantation, as recently supported, although its activity seems to be down-regulated after transplantation in patients who do not experience recurrence of the disease.

[Development of a device allowing for the perfusion and preservation of rat kidneys at -4 degrees C]. / Mise au point d'une machine permettant la perfusion et la conservation des reins de rat à -4 degrees C.

OBJECTIVES: In order to prolong the cold ischaemia time and to improve the quality of donor kidneys, we have designed and developed a renal perfusion machine allowing the control of perfusion parameters (temperature, pressure, flow rate, resistance) during the various phases of a kidney perfusion and storage protocol at -4 degrees C. ANIMALS, MATERIALS AND METHODS: Twenty four rat kidneys were removed and the effects of perfusion and storage at -4 degrees C were studied using a perfusion/storage machine allowing the controlled addition of 2,3 butanediol in University of Wisconsin (UW solution). The kidneys were stored for 96 hours at -4 degrees C and were studied in terms of perfusion parameters (pressure, resistance) and according to their histological appearance. RESULTS: The machine allows controlled perfusion of a cryoprotective agent and preservation of kidneys at -4 degrees C for 96 hours. CONCLUSION: In animals, it is possible to store kidneys at a temperature of -4 degrees C for 96 hours by using a vecor solution (UW solution) and a cryoprotective agent (2,3 butanediol). The perfusion and storage of organs under these conditions must be performed by a computer-assisted machine, allowing monitoring and control of the various steps of perfusion/storage.

[Evaluation of a new technique of extracorporeal perfusion of the swine liver on a swine-primate model]. / Evaluation d'une nouvelle technique de perfusion extracorporelle de foie de porc sur un modèle porc-primate.

With the increasing success of liver transplantation there is an urgent need for developing an artificial liver support system to be used in patients with liver failure. An extracorporeal porcine liver perfusion machine was successfully tested in animals with experimental liver failure. Livers were flushed, removed from 35 kg pigs and placed in a heated sterile cassette. The portal vein and the hepatic artery of the graft were connected to the arterial system of the animals. The perfusion pressure of the hepatic artery was regulated via a pressure-flow computerized feed-back device. The venous flow was reinfused from the hepatic veins of the graft to the jugular vein of the animals. The experimental work consisted in two steps: 1. evaluation of clinical and biological consequences of liver perfusion in healthy animals (Group A = pigs, n = 3; group B = primates, n = 3); 2. evaluation of the efficiency of the liver perfusion in animals with ischemic liver failure (Groupe D = pigs, n = 6). The control group (Group C = pigs, n = 7) consisted of pigs with ischemic liver failure without hepatic support. No major clinical or biological adverse effects are reported in groups A and B excepted a thrombocytmia and a marked increase in serum transaminases levels in group B. Liver function as assessed by the bile flow was good in both groups. Comatose pigs with ischemic hepatic failure (group D) recovered a subnormal neurological status in five out of six cases. Serum ammoniemia level were significantly decreased (from 1076 +/- 163 to 255 +/- 32 umol/l). A decrease in serum bilirubine levels and an improvement in the coagulation profile were observed in the perfused animals. Pigs and primates tolerated the perfusion procedure well and beneficial effects were observed in perfused pigs with experimental liver failure.