Two case reports of generalized pustular psoriasis of pregnancy: Different outcomes.

Generalized pustular psoriasis of pregnancy is a rare dermatosis with potential serious consequences for both the mother and fetus. Treatment is difficult and historically steroids were the mainstay of treatment. Cyclosporin has been used for a few cases resistant to steroids. We report our own experience of two cases of generalized pustular psoriasis of pregnancy. Cases of generalized pustular psoriasis of pregnancy need review by a dermatologist with experience of skin disorders in pregnancy. Both the fetus and mother need to be monitored closely when systemic illness occurs, as there is a risk of stillbirth. Maternal sepsis is a known complication of generalized pustular psoriasis of pregnancy. Cyclosporin, when used appropriately is effective and relatively safe.

Molecular analysis of successful cell line selection in transfected GS-NS0 myeloma cells.

The production of recombinant proteins from mammalian cells is now an essential part of biotechnology. However, despite this importance, the detailed characteristics of good producing cell lines remain largely unknown. The industrially important GS-NS0 mammalian expression system is able to produce large amounts of protein from relatively few copies of recombinant genes. This makes GS-NS0 cell lines ideal candidates to study the consequence of recombinant plasmid transfection in mammalian cells. This study investigated the molecular features of a panel of 17 randomly chosen GS-NS0 cell lines engineered to produce a recombinant antibody. The research analysed antibody production via enzyme-linked immunosorbent assay (ELISA), and investigated the molecular features of the transfectants by Northern, Southern and copy number analysis. The cell lines generated produced a range of antibody concentrations. In addition, for transfectants defined as producers of recombinant antibody there was a positive correlation between specific productivity and heavy chain mRNA expression. The use of Northern and Southern analysis allowed determination of the functional integrity of the transfected plasmid. Over 50% of the transfectants studied had molecular defects at the level of mRNA and/or cDNA. Cell lines were identified with suspected defects in the regulatory regions of transfected genes in addition to cell lines which lacked recombinant genes. Also, "false-positive" cell lines were generated which were able to overcome the GS selection pressure without producing any recombinant antibody. This article discusses these findings in relation to vector design.

Increased protein expression of matrix metalloproteinases -1, -3, and -9 and TIMP-1 in patients with gluten-sensitive enteropathy.

Gluten-sensitive enteropathy is characterized by small intestinal damage. The pathogenic mechanisms involved are not precisely understood. There is recent interest in the possibility that matrix metalloproteinases might play a pathogenic role. Using immunohistochemistry technique, we examined the protein expression of matrix metalloproteinases-1, -3, and -9 and the tissue inhibitor metalloproteinase-1 in duodenal biopsies from 30 patients with celiac disease and dermatitis herpetiformis. We demonstrated that the percentage of cells expressing these enzymes and their inhibitor in all patients was significantly greater than in the normal controls (P < 0.0001). This was evident even in patients with a minimal lesion but was most marked in patients with severe damage, mirroring the degree of inflammation in the small intestinal tissue. The increased expression of these enzymes and their inhibitor in the duodenal mucosa of patients with gluten-sensitive enteropathy suggests a role for these enzymes in the tissue remodeling which is a feature of these disorders.

Mammalian cell factories for efficient and stable protein expression.

As the commercial market for therapeutic protein production from mammalian cells has expanded, so has the requirement for improved efficiency and stability of production. Rapid developments have been made in understanding the molecular environment of transgenes in chromatin, including elucidation of the contribution of epigenetic modifications to expression, and this understanding is being used to enhance expression from host cells. Technical advances surrounding the 'omics' revolution are enabling the rational identification of complex control factors that define the flow of information from transgene to desired protein. Using information from 'omics' interrogations, directed cell engineering has been employed to enhance the translational and secretory capacity of host cells. Taken together, these recent advances are likely to lead to improved routes for protein production in the future.

Phenotypic variation during cloning procedures: analysis of the growth behavior of clonal cell lines.

The production of recombinant protein from mammalian cells is a key feature of the biotechnology industry. However, the generation of recombinant mammalian cell lines is still largely performed on an empirical basis and there are many potential areas for enhancement. We have shown previously that despite two rounds of limiting dilution cloning (LDC) of recombinant cell lines, there remained a high degree of heterogeneity in the resulting cell lines. We suggested that a rapid phenotypic drift occurred with these cells. It was unclear if this was a consequence of the added burden of production of a recombinant protein, the selection procedures, or merely an inherent feature of cell growth in culture. To address this, we have subjected untransfected (parental) cells to three successive rounds of LDC and monitored the growth properties of the resultant cells. The results show that despite repeated rounds of cloning, it was not possible to obtain phenotypically similar cell lines. We also demonstrated that this phenotypic drift is not due to gross changes in the protein p27, a key regulators of the cell cycle. Although cells with a range of growth properties were observed even after three rounds of cloning, the variation in growth patterns between cell lines decreased after cloning. Hence, we suggest that by cloning it may be possible to generate untransfected cells, which have particular growth properties. Starting with a well-defined population of parental cells may aid in the subsequent generation of tranfectants with desired growth properties.

Cost analysis of narrowband UVB phototherapy in psoriasis.

BACKGROUND: There are few data available to health care providers regarding the costs of treating patients with psoriasis, and specifically the cost of phototherapy. OBJECTIVES: As narrowband UVB (TL-01) has now become an established therapy for patients with psoriasis requiring phototherapy, we determined the annual cost of delivering TL-01 treatment in a university hospital. METHODS: The costing evaluation was from a hospital perspective and the strategy used was a microcosting detailed collection of resources used. RESULTS: The annual cost of TL-01 treatment in our teaching hospital was 53,555.00 euros. Staffing accounted for 70% of the cost. The average individual costs were 325.00 euros (range: 57.20-972.40). CONCLUSION: These costs are significant but remain less expensive than inpatient treatment.

Molecular definition of predictive indicators of stable protein expression in recombinant NS0 myeloma cells.

We have generated a molecular description of the loci at which stability/instability of expression of a monoclonal antibody (MAb) (anti-CD38) occurs within the GS-NS0 expression system. Critically, these data show that, in the absence of changes to copy number for the recombinant gene sequences, all cell lines examined exhibit a progressive loss (instability) in expression of mRNA during prolonged culture. However, not all cell lines express instability at the level of MAb protein production. The molecular distinction between stable and unstable production at the protein level is a reflection of the cellular amount of recombinant mRNA encoding MAb. Our data indicate a threshold level, a putative saturation point for utilisation of mRNA in translational/secretory events, that defines stability or instability of protein production. Above this level of recombinant mRNA expression, cell lines are stable, whereas below this level cell lines will show instability of protein production. Our studies indicate that absolute levels of expression of recombinant mRNA encoding for MAb in the GS-NS0 expression system offer a potential predictive indicator for the selection of stable cell lines for scale-up. These studies identify molecular facets of host cell biology of generic interest for gene regulation and expression and define techniques and approaches for enhancement of recombinant protein expression and process development.

Stability of recombinant protein production in the GS-NS0 expression system is unaffected by cryopreservation.

Mammalian cells form a very important part of the repertoire of production systems available to scientists involved in the production of recombinant proteins. During the production of therapeutic proteins it is vital for regulatory approval of products that no phenotypic or genetic changes are observed in the cell line or product. As part of the generation and development of therapeutic protein production, cell lines have to be frozen at various stages to create cell banks. If cryopreservation and revival of frozen stocks were to give rise to any phenotypic changes in the cells, this would again be detrimental to the further development of that particular cell line. This study uses one of the most industrially important expression systems, the GS-NS0 expression system, to examine the effect of cryopreservation on the growth and productivity profile of cell lines that exhibit differential degrees of stability during prolonged (production) culture periods. Results show that cryopreservation and revival procedures do not alter the stability characteristics of cell lines. This type of information is of great value in definition of protocols for cell line development.

Stability of protein production from recombinant mammalian cells.

One of the most important criteria for successful generation of a therapeutic protein from a recombinant cell is to obtain a cell line that maintains stability of production. If this is not achieved it can generate problems for process yields, effective use of time and money, and for regulatory approval of products. However, selection of a cell line that sustains stability of production over the required time period may be difficult to achieve during development of a therapeutic protein. There are several studies in the literature that have reported on the instability of protein production from recombinant cell lines. The causes of instability of production are varied and, in many cases, the exact molecular mechanisms are unknown. The production of proteins by cells is modulated by molecular events at levels ranging from transcription, posttranscriptional processing, translation, posttranslational processing, to secretion. There is potential for regulation of stability of protein production at many or all of these stages. In this study we review published information on stability of protein production for three industrially important cell lines: hybridoma, Chinese hamster ovary (CHO), and nonsecreting (NS0) myeloma cell lines. We highlight the most likely molecular loci at which instability may be engendered and indicate other areas of protein production that may affect stability from mammalian cells. We also outline approaches that could help to overcome the problems associated with unpredictable expression levels and maximized production, and indicate the consequences these might have for stability of production.