Optimizing the transient transfection process of HEK-293 suspension cells for protein production by nucleotide ratio monitoring.

Large scale, transient gene expression (TGE) is highly dependent of the physiological status of a cell line. Therefore, intracellular nucleotide pools and ratios were used for identifying and monitoring the optimal status of a suspension cell line used for TGE. The transfection efficiency upon polyethyleneimine (PEI)-mediated transient gene delivery into HEK-293 cells cultured in suspension was investigated to understand the effect of different culture and transfection conditions as well as the significance of the culture age and the quality of the cell line used. Based on two different bicistronic model plasmids expressing the human erythropoietin gene (rHuEPO) in the first position and green fluorescent protein as reporter gene in the second position and vice versa, a completely serum-free transient transfection process was established. The process makes use of a 1:1 mixture of a special calcium-free DMEM and the FreeStyle™ 293 Expression Medium. Maximum transfectability was achieved by adjusting the ratio for complex formation to one mass part of DNA and three parts of PEI corresponding to an N/P (nitrogen residues/DNA phosphates) ratio of 23 representing a minimum amount of DNA for the polycation-mediated gene delivery. Applying this method, maximum transfectabilities between 70 and 96 % and a rHuEPO concentration of 1.6 µg mL(-1) 72 h post transfection were reached, when rHuEPO gene was expressed from the first position of the bicistronic mRNA. This corresponded to 10 % of the total protein concentration in the cell-free supernatant of the cultures in protein-free medium. Up to 30 % higher transfectabilities were found for cells of early passages compared to those from late passages under protein-free culture conditions. In contrast, when the same cells were propagated in serum-containing medium, higher transfectabilities were found for late-passage cells, while up to 40 % lower transfectabilities were observed for early-passage cells. Nucleotide pools were measured during all cell cultivations and the nucleoside triphosphate/uridine ratios were calculated. These 'nucleotide ratios' changed in an age-dependent manner and could be used to distinguish early- from late-passage cells. The observed effects were also dependent on the presence of serum in the culture. Nucleotide ratios were shown being applied to investigate the optimal passage number of cultured cell lines for achieving a maximum productivity in cultures used for transient gene expression. Furthermore, these nucleotide ratios proved to be different for transfected and untransfected cells, providing a high potential tool to monitor the status of transfection under various culture conditions.

Time course of T-cell responses to MOG and MBP in patients with clinically isolated syndromes.

CD4(+) T-cell lines (TCLs) from patients with clinically isolated syndromes (CIS) were selected with purified human myelin basic protein (MBP) and recombinant human myelin oligodendrocyte glycoprotein (rhMOG), at onset of neurological symptoms and when patients developed clinically definite multiple sclerosis (CDMS). The epitope specificity of each TCL was mapped with overlapping synthetic peptides. TCLs were assessed for their ability to secrete IFN-gamma, IL-4, and IL-6. Diverse patterns of epitope recognition were observed: (a) recognition of a broad spectrum of MBP peptide epitopes with evidence of shifts over time; (b) an initial T-cell response focused to a restricted segment of the MBP molecule (83-102) that broadened over the course of disease; and (c) persistence of a focused anti-MOG T-cell response. CIS patients who failed to develop CDMS maintained a focused epitope response against two to six MBP epitopes. Most MBP peptide-specific TCLs secreted considerable amounts of IFN-gamma and low amounts of IL-4 and IL-6, whereas anti rhMOG(Igd) peptide-specific TCLs secreted preferentially IL-4 and IL-6. These data raise important issues for the pathogenesis and treatment of multiple sclerosis (MS).

Oligoclonal bands and antibody responses in multiple sclerosis.

Multiple Sclerosis (MS) is an inflammatory disease of the Central Nervous System with multifocal areas of demyelination. Although its etiology and pathogenesis remain controversial, several lines of evidence indicate that MS is mediated by a misdirected immune response against one or several myelin proteins. The involvement of diverse leukocyte subsets and their products in MS is still the subject of considerable debate. The emphasis on T cells has derived mainly from the detection of activated T cells in MS plaques and analogies with animal models of MS. Because of these observations cell-mediated immunity has dominated the research field of MS to this day. However, in recent years the role of B cells, plasma cells and immunoglobulins in MS have been re-examined, and current findings indicate that humoral immunity also plays a major role in disease pathogenesis. B cells and their products could exert several potential effects during the course of MS. Firstly, autoantibodies against specific myelin antigens could mediate damage to myelin membranes. Secondly, some studies suggest that natural autoantibodies could enhance remyelination. Thirdly, antibodies directed against myelin components can participate in anti-idiotypic networks, which may regulate the course of MS. Increased intrathecal immunoglobulin synthesis reflected by raised IgG indices and an oligoclonal pattern is the most common abnormality detected in MS patients. The introduction of more sensitive procedures for protein detection has allowed demonstrating oligoclonal bands (OCBs) in up to 95 % of patients with clinically definite MS. Although the presence of OCBs in CSF of MS patients is now well established as a sensitive laboratory test to support the clinical diagnosis, OCBs may be present in other disorders, including those not directly related to infection or abnormal immune response. Nevertheless, the pathogenesis of OCBs in MS is still obscure, and despite extensive research their antigenic target(s) have yet to be established. Therefore, a critical task is to identify the specificity of such target(s), thus providing significant clues about MS etiology. For this purpose, novel molecular immunologic strategies have been recently developed to offer alternative approaches to identify putative antigens recognized by antibodies present in MS patients. The elucidation of the mechanisms and target(s) responsible for the onset of MS has obvious implications for the further development of specific therapies.