Status and transcriptional activity of a bovine-papillomavirus-I-based expression vector in a recombinant production cell line.

We have analysed the status and transcriptional activity of the bovine papillomavirus-I (complete BPV-I genome)-based expression vector pCES in CI27i-cell-line-derived 3TI cells used for the industrial production of recombinant human erythropoietin (rhuEpo). Complete tandem head-to-tail integration of about 600 vector copies at a single site of the cellular genome was observed. Deletions, insertions or rearrangements of pCES-specific sequences or extrachromosomal copies of vector sequences were not detected. Transcriptional analyses demonstrated that rhuEpo was abundantly expressed. BPV-I early transcription was detected, as expected from a BPV-I-transformed cell line; however, compared with the mouse metallothionein-I-promoter-driven rhuEpo-specific transcription, it was very weak. Late BPV-I transcription was also not detected in 3TI cells under conditions of large-scale rhuEpo production. Therefore this expression system proved to be safe and suitable for the production of rhuEpo.

Repeated batch cultivation of rBHK cells on Cytodex 3 microcarriers: antithrombin III, amino acid, and fatty acid metabolic quotients.

Anchorage-dependent human antithrombin III-producing recombinant baby hamster kidney (rBHK) cells were cultivated on Cytodex 3 microcarriers in repeated batch mode. During a 3-month experiment four different low-serum (0.025% fetal bovine serum) or serum-free medium formulations were evaluated for (a) the initial growth phase of cells and (b) the subsequent production phase, whereby two free fatty acid (FFA) supplements were examined with respect to their growth-promoting and product-formation-enhancing properties. Selected nutrient and (by)product consumption and production rates (including those for antithrombin III, amino acids, and fatty acids) are reported. The calculated metabolic quotients reflect the prevailing slow growth conditions (mu approx. 0.06 day-1) associated with microcarrier cultures. Specific antithrombin III productivities vary significantly as a function of the feed medium supplementation with FFA.

Inactivation of recombinant plasmid DNA from a human erythropoietin-producing mouse cell line grown on a large scale.

Experiments were carried out to assess the survival of recombinant plasmid DNA during large-scale production of recombinant human erythropoietin (rhuEPO) in a fermentation pilot plant. The analyses revealed DNA-degrading activities in the fermentation broth and in the waste-water, leading to rapid destruction of plasmid DNA added to medium or waste-water. The capability of the plasmid-DNA-spiked samples to transform competent bacteria was drastically reduced. The DNA-degrading activity in the waste-waters could be blocked by addition of EDTA or by boiling, indicating the presence of DNA-degrading enzymes (DNases). No plasmid-specific DNA sequences were detected in waste-water samples by in-vitro amplification with Taq-polymerase. Genomic DNA preparations of cell debris collected from waste-water samples only contained degraded plasmid DNA. Furthermore, it was shown that intact plasmid DNA could be degraded to fragments of less than 1000 bp by incubation at 121 degrees C for 20 min, leading to a decrease in the plasmid-specific transforming capacity by a factor of 10(3) per minute. Thus, DNA from the rhuEPO production pilot plant was efficiently inactivated at three different levels: (i) in the fermentation medium (DNase), (ii) in the waste-water container (DNase), and (iii) by heat inactivation for 20 min at 120 degrees C. These results indicate that the probability of delivery of recombinant DNA into the environment is extremely low in such biotechnological production processes.

Effect of free fatty acids and phospholipids on growth of and product formation by recombinant baby hamster kidney (rBHK) and Chinese hamster ovary (rCHO) cells in culture.

Recombinant BHK and CHO cells producing human antithrombin III (rh ATIII) were used to investigate the utilization of phospholipids and free fatty acids from low-serum (0.1% FBS) culture medium. Both cell lines show distinctly different patterns of fatty acid utilization. For rBHK ATIII cells it is shown that under low serum conditions several different combinations of free fatty acids (bound to bovine albumin) elicit an identical growth stimulatory effect although individual consumption and production rates of fatty acids are different. Increased fatty acid concentrations lead to increased uptake rates without any further effect on growth rate being observed. Recombinant antithrombin III formation is found to be a function of combinations and concentrations of fatty acids present in the culture medium.

Pitfalls of single radial immunodiffusion techniques used for the quantification of influenza haemagglutinin.

In the case of Tween-ether split vaccines the quantification of haemagglutinin is not achievable by the single radial immunodiffusion test alone. Aggregate formation of solubilized haemagglutinin frequently occurs when the applied detergent is removed and, therefore, a physico-chemical method including an effective disaggregation procedure like SDS treatment in combination with PAGE is recommended.

Quantification of haemagglutinin of influenza Tween-ether split vaccines by immunodiffusion.

The haemagglutinin content of monovalent influenza whole virus and Tween-ether split vaccines derived therefrom, were assayed comparatively using single radial immunodiffusion (SRID, the only test recommended for influenza vaccines by the European Pharmacopoeia Commission), quantitative SDS-polyacrylamide gel electrophoresis and immunization of guinea pigs. If SRID was performed with split vaccines, reduced haemagglutinin values were consistently recorded which were 50-25% of values obtained before disruption of virions. If, however, disruption was conducted in the presence of excess detergent thus preventing aggregate formation of solubilized haemagglutinin, test values comparable to those of whole virus vaccines were obtained. In agreement with these results, immunization experiments revealed that whole virus and the corresponding split vaccines exhibited comparable immunogenicity in guinea pigs. From SDS-polyacrylamide gel electrophoresis and densitometer tracings obtained by scanning the gels after staining with either Coomassie Blue or fluorescein isothiocyanate-labelled concanavalin A it was calculated that about 90% of whole virus HA2 was recovered in Tween-ether split vaccines. From our experiments we conclude that precise quantification of solubilized haemagglutinin is not achievable by the single radial immunodiffusion test alone. Aggregate formation of solubilized haemagglutinin frequently occurs when the applied detergent is removed and, therefore, a physico-chemical method including an effective disaggregation procedure like SDS treatment in combination with PAGE is recommended.

The quantification of the haemagglutinin content of influenza whole virus and Tween-ether split vaccines.

Monovalent whole virus and Tween-ether split vaccines prepared from influenza A/Bangkok, A/Brazil and B/Singapore were assayed for haemagglutinin content using single radial immunodiffusion (SRID), quantitative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunization of guinea pigs. When SRID was performed with split vaccines, haemagglutinin values were consistently recorded which were in the range of 50 to 25% of the values obtained before disruption of virions. When, however, disruption was conducted in the presence of excess detergent, thus preventing aggregate formation of solubilized haemagglutinin, test values comparable with those of whole virus vaccines were obtained. In agreement with these results, immunization experiments revealed that whole virus and corresponding split vaccines exhibited comparable immunogenicity in guinea pigs. Additionally it could be calculated from SDS-PAGE and densitometer tracings, obtained by scanning the gels after staining with either Coomassie blue or FITC-Con A, that 90 to 95% of whole virus HA2 was recovered in Tween-ether split vaccines. On the basis of these findings we conclude that precise quantification of Tween-ether split vaccines is not possible by the SRID test alone. As aggregate formation of solubilized haemagglutinin occurs, we suggest that either a physico-chemical method including a disaggregation procedure, such as SDS treatment, or immunological evaluation of the original whole virus preparation before disruption of virions should be applied as an additional criterion for quantification of influenza Tween-ether split vaccines.

Blood transfusion-induced suppression of cellular immunity in man.

The effect of planned blood transfusions on cell-mediated immunity was studied in previously nontransfused prospective kidney graft recipients. Following transfusion of washed erythrocytes a marked suppression of cellular immunity was found, indicated by reduced response to mitogenic (PHA, Con A, PWM) and antigenic stimulation (Ag-C containing PPD, tetanus toxoid, streptolysin, mumps, vaccinia antigen). A second transfusion led to a more pronounced and prolonged immunosuppression. No suppression was found when autologous blood was applied to volunteers. Preliminary results show autologous and allogeneic MLR suppression when mitomycin-C treated patient cells taken after transfusion are added. Our findings indicate that blood transfusion-induced suppression of cell-mediated immunity might be caused by an unspecific suppressor cell.

[New placental protein (PP15) with immunosuppressive properties (author's transl)]. / Neues Plazentaprotein (PP15) mit immunsuppressiver Wirkung.

The isolation and characterization of a new placental protein (PP15) which has immunosuppressive properties are described. The protein was purified from extracts of human term placentae by rivanol and ammonium sulfate fractionation, gel filtration, ion exchange chromatography, preparative zone electrophoresis, and chromatography on hydroxyapatite. PP15 was found to have a sedimentation coèfficient of 2.9 S and a molecular weight of 30,700 daltons as determined by ultracentrifugation; its molecules apparently are composed of two identical subunits which are held together by non-covalent bonds. The electrophorectic mobility of PP15 corresponds to that of albumin. PP15 is a glycoprotein and contains 3.3% carbohydrates (hexoses 2.8%, hexosamines 0.3%, sialic acid 0.2%). The amino acid composition of this protein was also determined: the most abundant amino acids in the peptide chain were found to be glutamic acid, aspartic acid, isoleucine, and leucine. PP15 has immunosuprressive properties: on testing its effect on the lymphoycte transformation in the MLC-test in vitro a significant inhibitory activity could be demonstrated.

Evidence for subtypic determinants in the HLA-DW3 cluster.

This study was undertaken to get more insight into the previously suggested heterogeneity of the HLA--DW3 cluster. Preliminary evidence of DW3 heterogeneity was derived from results of intrafamilial mixed lymphocyte culture tests (MLC) where cells of apparently homozygous offspring revealed unexpected stimulations of one of the parents' cells. Therefore, 15 different homozygous typing cells (HTCs) of DW3 specificity were tested against 43 HLA-DW3 heterozygous individuals. The response patterns of the 43 HLA-DW3 heterozygous cells toward 13 HTCs leads to the definition of at least three groups of DW3 stimulating cells. According to these patterns, four groups of responding cells could be classified. These results were confirmed by a MLC checkerboard experiment running all DW3-HTCs against each other. Discussing all possible explanations for these observations, the authors conclude that the existence of DW3 subtypes having some properties in common is the most likely interpretation of the results obtained. Family segregation studies will be needed to define the genotypic situation of the DW3 cluster.