The Rotary Cell Culture System (RCCS) is a new technology for growing anchorage dependent or suspension cells in the laboratory. The RCCS is a horizontally rotated, bubble free disposable culture vessel with diffusion gas exchange. The system provides a reproducible, complex 3D in vitro culture system with large cell masses. During cell growing the rotation speed can be adjusted to compensate for increased sedimentation rates. The unique environment of low shear forces, high mass transfer, and microgravity, provides very good cultivating conditions for many cell types, cell aggregates or tissue particles in a standard tissue culture laboratory. The system enables to culture HepG2 cells on Cytodex 3 microcarriers (mcs) to high densities. We inoculated 2 x 10(5)/ml HepG2 cells and 200 mg Cytodex 3 mcs in 50 ml Williams E medium (incl. 10% FCS) allowing them to attach to the mcs in the rotating vessel (rotation rate 14-20 rpm). HepG2 cells readily attached to the mcs while the vessel was rotating. Attachment of HepG2 to the mcs was about 50% after 24 hrs and 100 % within 48 hrs. After 72 hrs of rotary culturing small aggregates of Hep G2 on mcs were built. HepG2 cells and the aggregates rotated with the vessel and did not settle within the vessel or collide with the wall of the vessel. We conclude that this new RCCS is an excellent technology for culturing HepG2 cells on Cytodex 3 mcs. The system is easy to handle and enables to culture anchorage dependent cells to high densities in a short period.

On-line HDF treatment can routinely be provided with ultra-pure dialysis fluids and sterile substitution fluids at pyrogen-free levels. The online preparation of substitution fluids thus can be considered microbiologically safe.

Administration of adequate amounts of commercial infusion fluids renders modern convective dialysis modalities, such as hemodiafiltration, labor-intensive and costly. Preparation of infusate by cold sterilization of dialysis fluid, which is abundantly available, and its immediate (online) use, in contrast, enables a large volume fluid exchange in a cost-effective manner. Recent developments aimed at more hygienic and user-friendly online systems with increased operational flexibility. As a result the novel ONLINEplus system does not only provide online prepared infusate for convective dialysis therapy, but also for priming and rinsing of the extracorporeal blood circuit, for intradialytic bolus administration, and for re-infusion of patients' blood as well. Production of infusate from potentially impure dialysis fluid containing endotoxins and other pyrogens raises severe concerns of affecting the patients' well-being. To assess its safety, the online system was challenged with microbially contaminated dialysis fluid. Despite high levels of microbial counts (7.5 x 104 +/- 105 CFU/ml), endotoxin concentration (14.1 +/- 7.7 IU/ml and 9.265 +/- 3.000 IU/ml, as measured turbidimetrically and chromogenically, respectively) and cytokine-inducing activity (20,827 +/- 3,082 pg IL-1Ra/Mio WBC), we failed to detect contaminants in the final infusate during a 5 week laboratory testing period. In addition, infusate samples complied consistently with the European Pharmacopeia test for sterility. The present online system is comprehensive, operates user-friendly, and provides microbiologically safe infusate in large quantities. In this way, both patients and dialysis staff will benefit from improved dialysis therapy and reduced treatment-related labor burden, respectively. Moreover, convective dialysis modalities will become less expensive.

End-stage renal disease (ESRD) patients are known to suffer from chronic inflammation as the result of an ongoing subacute cytokine induction, which may contribute considerably to dialysis-related, long-term morbidity and mortality. Preparation of infusate from cytokine-inducing dialysis fluid and its administration in large quantities as well as the use of high-flux membranes bear the risk of aggravating the chronic inflammatory response among online hemodiafiltration (online HDF) patients. In order to assess the inflammatory risk associated with online HDF, we compared the cytokine induction profile of ESRD patients receiving either online HDF or low-flux hemodialysis (low-flux HD). Specifically, we measured spontaneous and lipopolysaccharide (LPS)-stimulated tumor necrosis factor alpha (TNFalpha) and interleukin-1 receptor antagonist (IL-1Ra) release during ex vivo incubation of whole blood. Ultrapure dialysis fluid and polysulfone membranes were used for both treatment modalities. LPS-stimulated release of TNFalpha and IL-1Ra was elevated for both online HDF and low-flux HD patients compared to healthy individuals (TNFalpha: 2,336 +/- 346 and 2,192 +/- 398 versus 1,218 +/- 224 pg/106 white blood cells [WBC]; IL-1Ra: 2,410 +/- 284 and 2,326 +/- 186 versus 1,678 +/- 219 pg/106 WBC). Likewise, spontaneous production of TNFalpha, but not IL-1Ra, was higher in online HDF and low-flux HD patients than in normal controls (37 +/- 32 and 22 +/- 19 versus 0.8 +/- 0.3 pg TNFalpha/106 WBC). There was no difference in spontaneous and LPS-stimulated cytokine release between both dialysis groups. In addition, intradialytic cytokine induction was not significant for either treatment modality as spontaneous and LPS-stimulated cytokine release were not increased postdialysis. These findings indicate that online HDF does not contribute to chronic leukocyte activation and, consequently, does not place ESRD patients at greater risk with respect to inflammatory morbidity and mortality.

Processing of the transfer operon mRNA of the conjugative resistance plasmid R1-19 results in the accumulation of stable traA mRNAs. The stable traA transcripts found in vivo have identical 3' ends within downstream traL sequences, but vary at their 5' ends. The 3' ends determined coincide with the 3' base of a predicted large clover-leaf-like RNA secondary structure. Here we demonstrate that this putative RNA structure, although part of a coding sequences, stabilizes the upstream traA mRNA very efficiently. We also show that the 3' ends of the stable mRNAs are formed posttranscriptionally and not by transcription termination. Half-life determinations reveal the same half-lives of 13 +/- 2 min for the traA mRNAs transcribed from hybrid lac-traAL-cat test plasmids, the R1-19 plasmid, and the F plasmid. Protein expression experiments demonstrate that the processed stable traA mRNA is translationally active. Partial deletions of sequences corresponding to the predicted secondary structure within the traL coding region drastically reduce the chemical and functional half-life of the traA mRNA. The results presented here unambiguously demonstrate that the proposed secondary structure acts as an efficient intracistronic mRNA stabilizer.