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Biotechnological production of recombinant proteins for human therapy requires a cultivation of the host organism in a nutrient medium free of animal substances. Therefore, various nutrient media for the new expression system Leishmania tarentolae were developed and examined according to their cultivation conditions as static suspension culture and agitated culture. Investigations resulted in the development of a serum-free but hemin containing medium, based on yeast extract and buffer salts. Here we report that a high and stable specific growth rate of 0.103 h(-1) and a maximal cell density of 1 x 10(9) cells ml(-1) is obtained in an alternative medium, the YE-medium. For the newly developed medium, the successful expression of enhanced green fluorescent protein and the adaptation of the cultivation from the agitated culture to the bioreactor could be shown. Furthermore, an analytical method for detection of the essential, organic iron source hemin was established. The consumption of hemin was monitored because hemin is a potentially important process parameter for bioprocess control. With knowledge of these results, an improved expression system is available as an alternative to commonly used cell cultures for the production of recombinant proteins.

The aim of this study was to evaluate in vitro and in vivo imaging of HER-2-over-expressing tumours using near-infrared optical imaging. A fluorochrome probe was designed by coupling Cy5.5 to anti-HER-2 antibodies. Cells over-expressing (SK-BR-3 cells) or normally expressing (PE/CA-PJ34 cells) the HER-2 protein were incubated with the probe. After removing unbound probe molecules, fluorescence intensities were determined (a.u.: arbitrary units). Cells were additionally investigated using FACS and laser scanning microscopy. The probe was also injected intravenously into tumours bearing SK-BR-3 ( n=3) or PE/CA-PJ34 ( n=3). Whole-body fluorescence images were generated and analysed. The incubation of SK-BR-3 cells with the probe led to higher fluorescence intensities [2,133 (+/-143) a.u.] compared to controls [975 (+/-95) a.u.]. The results from FACS and immunocytochemical analysis were in agreement with these findings. A distinct dependency between the fluorescence intensity and the cell number used in the incubations was detected. In vivo, the relative fluorescence intensities in SK-BR-3 tumours were higher than in PE/CA-PJ34 tumours at 16-24 h after probe application. HER-2-over-expressing tumours were depictable in their original size. Labelling of HER-2 with Cy5.5 is suitable for in vitro and in vivo detection of HER-2-over-expressing tumour cells.

One approach to the tissue engineering of vascular structures is to develop in vitro conditions in order ultimately to fabricate functional vascular tissues before final implantation. In our experiment, we aimed to develop a new combined cell seeding and perfusion system that provides sterile conditions during cell seeding and biomechanical stimuli in order to fabricate autologous human vascular tissue in vitro. The cell seeding and perfusion system is made of Plexiglas and is completely transparent (Berlin Heart, Berlin, Germany; University Hospital Benjamin Franklin, Berlin, Germany). The whole system consists of a cell seeding chamber that can be incorporated into the perfusion system and an air-driven respirator pump connected to the bioreactor. The cell culture medium continuously circulates through a closed-loop system. We thus developed a cell seeding device for static and dynamic seeding of vascular cells onto a polymeric vascular scaffold and a closed-loop perfused bioreactor for long-term vascular conditioning. The cell seeding chamber can be easily connected to the bioreactor, which combines continuous, pulsatile perfusion and mechanical stimulation to the tissue-engineered conduit. Adjusting the stroke volume, the stroke rate, and the inspiration/expiration time of the ventilator allows various pulsatile flows and different levels of pressure. The whole system is a highly isolated cell culture setting, which provides a high level of sterility and a gas supply and fits into a standard humidified incubator. The device can be sterilized by ethylene oxide and assembled with a standard screwdriver. Our newly developed combination of a cell seeding and conditioning device provides sterile conditions and biodynamic stimuli for controlled tissue development and in vitro conditioning of an autologous tissue-engineered vessel.

We describe a novel membrane surface display system that allows the anchoring of foreign proteins in the cytoplasmic membrane (CM) of stable, cell wall-less L-form cells of Escherichia coli and Proteus mirabilis. The reporter protein, staphylokinase (Sak), was fused to transmembrane domains of integral membrane proteins from E. coli (lactose permease LacY, preprotein translocase SecY) and P. mirabilis (curved cell morphology protein CcmA). Both L-form strains overexpressed fusion proteins in amounts of 1 to 100 microg ml(-1), with higher expression for those with homologous anchor motifs. Various experimental approaches, e.g., cell fractionation, Percoll gradient purification, and solubilization of the CM, demonstrated that the fusion proteins are tightly bound to the CM and do not form aggregates. Trypsin digestion, as well as electron microscopy of immunogold-labeled replicas, confirmed that the protein was localized on the outside surface. The displayed Sak showed functional activity, indicating correct folding. This membrane surface display system features endotoxin-poor organisms and can provide a novel platform for numerous applications.

The purpose of this study was to evaluate the effect of patient position on the mass transfer area coefficient (KoA) and to characterize drain/fill profiles in an effort to enhance efficiency of automated peritoneal dialysis. Over 100 exchanges were performed in 38 stable peritoneal dialysis patients to either determine the small solute KoA in the supine versus upright position or to characterize fill/drain profiles. The KoA for all solutes tested was significantly greater in the supine position compared with the upright position (P < 0.05). Fill profiles revealed the fill rate to be a function of fill height (P < 0.001) and patient position (supine > upright [P < 0.001]). Analysis of drain flow rate versus time revealed an initial segment of high outflow (350 +/- 89 mL/min) followed by an abrupt transition to a segment characterized by slow drainage (36 +/- 21 mL/min). The first segment of drain only took 5.6 +/- 2.3 minutes (42% of the total drain time); in that time, 83% +/- 10% of the dialysate was drained. The transition volume (volume of dialysate remaining at the time the transition occurs, excluding residual volume) correlated with body surface area (R = 0.52, P < 0.01). In conclusion, automated peritoneal dialysis treatment (including intermittent peritoneal dialysis, which may be done in the upright position) should be done in the supine position to optimize the KoA, and shortening drain time to include only the initial segment of high outflow will improve the efficiency and convenience of therapy.

Background: The significance of hepatitis C in kidney transplant recipients is unclear. The prevalence of antibodies to hepatitis C among candidates for transplantation is up to 50% in some centers., Methods: We screened 640 frozen serum samples obtained pretransplantation from all kidney recipients at the Medical College of Wisconsin between January 1979 and March 1990 for antibody to hepatitis C using the second generation immunoassay. Charts were reviewed from all hepatitis C antibody-positive (anti-HCV+) patients and 256 randomly chosen hepatitis C antibody negative (anti-HCV-) controls. Actuarial patient and graft survival in these two groups were determined., Results: The prevalence of anti-HCV was 8.3%. Blacks and i.v. drug users were disproportionately represented in the anti-HCV+ group. Of the anti-HCV+ patients, 18.9% developed chronic hepatitis independent of race. Black anti-HCV+ patients had a 5-year graft survival of 28 +/- 11% compared to 67 +/- 7% in black anti-HCV- patients (P = 0.003). Black anti-HCV-, white anti-HCV-, and white anti-HCV+ patients all had similar graft survival. Anti-HCV was not a poor prognostic indicator for overall patient survival or the development of aplastic anemia and malignancies including hepatocellular carcinoma., Conclusions: Anti-HCV is a significant risk factor for reduced kidney graft survival in blacks apart from i.v. drug abuse. Black anti-HCV- patients had graft survival similar to white transplant recipients, indicating that anti-HCV may be one marker for the poorer graft survival in blacks that has been observed in most transplant programs. Anti-HCV in kidney transplant recipients increases the risk for the development of chronic hepatitis post-transplant.

The adequacy of the peritoneal dialysis prescription is of great concern. Although the use of urea kinetics has become the standard in hemodialysis, its usefulness in peritoneal dialysis is unclear. It has been suggested that creatinine clearance may correlate with clinical outcome in CAPD but, as with urea kinetics, its predictive value is not established. The efficacy number (EN), which only requires a four hour exchange, was introduced as a simpler approach to creatinine kinetics. These three kinetic models were correlated to clinical outcome in 18 stable CAPD patients over a 12 month study period. The patients were divided into three groups: good (G), intermediate (I), and poor (P) based on uremic symptoms, mortality, hospital days, biochemical indices and the need for transfer to hemodialysis. Both forms of creatinine kinetics (weekly creatinine clearance, EN) were able to differentiate between the G, I, and P outcome groups (P < 0.05). The weekly uea Kt/V was able to differentiate between the G and P groups (P < 0.05) but not between the I and the other two outcome groups. Both urea and creatinine kinetics predict clinical outcome in CAPD. However, creatinine kinetics may be a more sensitive predictor. The efficacy number was just as sensitive as creatinine clearance in predicting clinical outcome yet simpler to gather the data for its calculation.

Acute renal failure caused from an obstruction by the gravid uterus is a rare complication of pregnancy. Only 13 cases have been reported. We report a case of obstructive acute renal failure in a patient at 34 weeks' gestation with a twin pregnancy complicated by polyhydramnios. The serum creatinine peaked at 1,078 mumol/L (12.2 mg/dL), higher than in the previously reported patients. The striking feature of her clinical course was the immediate resolution of anuria following amniotomy, thus avoiding the need for dialysis, ureteral stenting, or immediate surgical delivery. This patient illustrates the potential importance of increased uterine pressure on the ureters as a cause of significant obstructive renal failure during pregnancy.

Patients with microscopic hematuria are generally referred for urologic investigation. We describe 30 patients with normal renal function referred to our clinic during the years 1970 through 1987 for evaluation of hematuria, usually microscopic, in whom prior urologic and radiological studies had failed to determine the cause of bleeding. Urinary sediment from the patients and first-degree relatives revealed hemoglobin and red blood cell casts; the inheritance pattern was consistent with autosomal dominant transmission. During follow-up for up to 18 years, renal function remained normal, thus confirming the diagnosis of benign familial hematuria. Immunoglobulin A nephropathy and Alport's syndrome were less common than benign familial hematuria and could be differentiated from it by history, physical examination, and routine laboratory testing. Since benign familial hematuria is a common disorder in adults with hematuria and normal renal function, urinary sediment from patients and family members should be examined before extensive urologic and radiological procedures are performed.