A rapid cell-based assay for determining poloxamer quality in CHO suspension cell culture.

Poloxamers are water-soluble polymers that are widely used in cell culture bioprocessing to protect cells against shearing forces. Use of poor-quality poloxamers may lead to a drastic reduction in cell growth, viabilities and productivities in cell culture-based manufacturing. In order to evaluate poloxamer quality and promote more consistent performance, a rapid cell membrane adhesion to hydrocarbon assay was developed based on the adhesive properties of cell membranes to selective hydrocarbons. The assay can identify a poor-performing poloxamer characterized by significant drop in viable cell density and percent viability. The assay was verified across multiple good and bad poloxamer lots, and the results were in agreement with established cell growth and high-performance liquid chromatography assays.

Comparison of the behavior of CHO cells during cultivation in 24-square deep well microplates and conventional shake flask systems.

In biopharmaceutical production, the optimization of cell culture media and supplementation is a vital element of process development. Optimization is usually achieved through the screening of multiple media, feed and feeding strategies. However, most screening is performed in shake flasks, which makes the screening process very time consuming and inefficient. The use of small scale culture systems for the screening process can aid in the ability to screen multiple formulations during process development. In order to assess the suitability of 24 deep well (24DW) plates with the Duetz sandwich-covers as a small scale culture system for process development, we have tested growth and production performance of CHO cells in 24DW plates and conventional shake flask cultures. Multiple studies were performed to assess well-to-well and plate-to-plate variability in 24DW plates. Additional studies were performed to determine the applicability of 24DW plates for cell culture medium and supplement screening in batch and fed batch processes. Cultures in 24DW plates exhibited similar kinetics in growth, viability and protein production to those cultured in shake flasks, suggesting that 24DW plates with Duetz sandwich-covers can be effectively used for high throughput cell culture screening.

Tyrosine phosphorylation of caveolin-2 at residue 27: differences in the spatial and temporal behavior of phospho-Cav-2 (pY19 and pY27).

Caveolin-2 is an accessory molecule and the binding partner of caveolin-1. Previously, we showed that c-Src expression leads to the tyrosine phosphorylation of Cav-2 at position 19. To further investigate the tyrosine phosphorylation of Cav-2, we have now generated a novel phospho-specific antibody directed against phospho-Cav-2 (pY27). Here, we show that Cav-2 is phosphorylated at both tyrosines 19 and 27. We reconstituted this phosphorylation event by recombinantly coexpressing c-Src and Cav-2. We generated a series of Cav-2 constructs harboring the mutation of each tyrosine to alanine, singly or in combination, i.e., Cav-2 Y19A, Y27A, and Y19A/Y27A. Recombinant expression of these mutants in Cos-7 cells demonstrated that neither tyrosine is the unique phosphorylation site, and that double mutation of tyrosines 19 and 27 to alanine abrogates Cav-2 tyrosine phosphorylation. Immunofluorescence analysis of NIH 3T3 cells revealed that the two tyrosine-phosphorylated forms of Cav-2 exhibited some distinct properties. Phospho-Cav-2 (pY19) is concentrated at cell edges and at cell-cell contacts, whereas phospho-Cav-2 (pY27) is distributed in a dotlike pattern throughout the cell surface and cytoplasm. Further functional analysis revealed that tyrosine phosphorylation of Cav-2 has no effect on its targeting to lipid rafts, but clearly disrupts the hetero-oligomerization of Cav-2 with Cav-1. In an attempt to identify upstream mediators, we investigated Cav-2 tyrosine phosphorylation in an endogenous setting. We found that in A431 cells, EGF stimulation is sufficient to induce Cav-2 phosphorylation at tyrosines 19 and 27. However, the behavior of the two phosphorylated forms of Cav-2 diverges upon EGF stimulation. First, phospho-Cav-2 (pY19) and phospho-Cav-2 (pY27) display different localization patterns. In addition, the temporal response to EGF stimulation appears to be different. Cav-2 is phosphorylated at tyrosine 19 in a rapid and transient fashion, whereas phosphorylation at tyrosine 27 is sustained over time. Three SH2 domain-containing proteins, c-Src, Nck, and Ras-GAP, were found to associate with Cav-2 in a phosphorylation-dependent manner. However, phosphorylation at tyrosine 27 appears to be more critical than phosphorylation at tyrosine 19 for this binding to occur. Taken together, these results suggest that, in addition to the common characteristics that these two sites appear to share, phospho-Cav-2 (pY19) and phospho-Cav-2 (pY27) may each possess a set of unique functional roles.

Down-regulation of IL-2 production in T lymphocytes by phosphorylated protein kinase A-RIIbeta.

The beta isoform of the type II regulatory subunit (RIIbeta) of protein kinase A suppresses CREB transcriptional activity and c-Fos production in T cells following activation via the TCR. Because CREB is an integral nuclear transcription factor for IL-2 production by T cells, we tested the hypothesis that RIIbeta down-regulates IL-2 expression and IL-2 production in T cells. Stable transfection of RIIbeta in Jurkat T cells led to an approximately 90% reduction in IL-2 mRNA and IL-2 protein following T cell activation. The inhibition of IL-2 production was associated with phosphorylation of the RIIbeta subunit at serine 114 (pRIIbeta) and localization of pRIIbeta in intranuclear clusters. A serine 114 phosphorylation-defective mutant, RIIbeta(S114A), did not form these intranuclear clusters as well as wild-type RIIbeta, and did not inhibit IL-2 mRNA and protein synthesis, indicating that serine 114 phosphorylation is required for both nuclear localization and down-regulation of IL-2 production by RIIbeta. In contrast to its effect on IL-2, RIIbeta induced constitutive up-regulation of CD154 mRNA and cell surface expression. Thus, pRIIbeta differentially regulates gene expression following T cell activation. Unexpectedly, we also found that stable overexpression of another protein kinase A regulatory subunit, RIalpha, had the opposite effect on IL-2 expression, causing a 3- to 4-fold increase in IL-2 production following stimulation. In summary, our data demonstrate a novel mechanism by which serine 114 phosphorylation and nuclear localization of RIIbeta controls the regulation of gene expression in T cells.

Performance improvement with a multidisciplinary clinical guideline for patients undergoing minimally invasive thoracic surgery.

BACKGROUND: A horizontally integrated multidisciplinary clinical guideline was created and implemented in 1999-2000 for minimally invasive thoracic surgery (MIS). Guideline elements included complete appropriate preoperative evaluation, minimally invasive surgery techniques, aggressive anesthetic management and absolute pain control, immediate extubation, abolition of "routine" laboratory and imaging investigations, and early and aggressive postoperative patient mobilization in an integrated, multidisciplinary postthoracotomy rehabilitation program. METHODS: In a retrospective controlled cohort study, data were collected for the 501 procedures performed on 311 patients (MIS group) from July 1, 2000, to June 30, 2001, and for 130 similar procedures performed on 90 similar patients under a standard general thoracic surgery ad hoc clinical program from July 1, 1998, to June 30, 1999. RESULTS: After implementation of the clinical guideline, services expanded with a 345% increase in case volume, a 40% reduction in cost, no adverse effects, and increased referring-physician and patient satisfaction. DISCUSSION: Significant performance improvement was realized by implementing a multidisciplinary clinical guideline for thoracic surgery that seamlessly integrated all facets of diagnosis, therapy, and rehabilitation.

Localization of phospho-beta-dystroglycan (pY892) to an intracellular vesicular compartment in cultured cells and skeletal muscle fibers in vivo.

beta-Dystroglycan is a ubiquitously expressed integral membrane protein that undergoes tyrosine phosphorylation in an adhesion-dependent manner. Tyrosine 892 is now thought to be the principal site for recognition by the c-Src tyrosine kinase; however, little is known about the regulation of this phosphorylation event in vivo. Here, we generated a novel monoclonal antibody probe that recognizes only tyrosine 892 phosphorylated beta-dystroglycan (pY892). We show that upon tyrosine phosphorylation, beta-dystroglycan undergoes a profound change in its sub-cellular localization (e.g., from the plasma membrane to an internal membrane compartment). One possibility is that the net negative charge at position 892 causes the redistribution of beta-dystroglycan to this intracellular vesicular location. In support of this notion, mutation of tyrosine 892 to glutamate (Y892E) is sufficient to drive this intracellular localization, while other point mutants (Y892F and Y892A) remain at the plasma membrane. Interestingly, our colocalization studies with endosomal markers (EEA1, transferrin, and transferrin receptor) suggest that these phospho-beta-dystroglycan containing internal vesicles represent a subset of recycling endosomes. At the level of these internal vesicular structures, we find that tyrosine phosphorylated beta-dystroglycan is colocalized with c-Src. In addition, we demonstrate that known ligands for alpha-dystroglycan, namely, agrin and laminin, are able to induce the tyrosine phosphorylation of beta-dystroglycan. Finally, we show that tyrosine phosphorylated beta-dystroglycan is also detectable in skeletal muscle tissue lysates and is localized to an internal vesicular membrane compartment in skeletal muscle fibers in vivo. The generation of a phospho-specific beta-dystroglycan (pY892) mAb probe provides a new powerful tool for dissecting the role of dystroglycan phosphorylation in normal cellular functioning and in the pathogenesis of muscular dystrophies.

Perforin and Fas in murine gammaherpesvirus-specific CD8(+) T cell control and morbidity.

The immune system uses both virus-specific T cells and B cells to control the acute and latent phases of respiratory infection with the murine gammaherpesvirus 68 (gammaHV-68). We sought to further define the important effector mechanisms for CD8(+) T cells. First, depletion of the CD4(+) T cells resulted in a failure of most animals to drive the virus into latency, although lytic virus in the lung was reduced by approximately 1000-fold from its peak. Second, the absence of either perforin or Fas alone had no impact on the ability to reduce titres of lytic virus in the lung. Further neutralization of IFN-gamma in CD4-depleted P(+/+), P(-/-) or Fas(-/-) mice had no effect. To define the requirements for Fas or perforin more clearly, two sets of chimeric mice were constructed differing in perforin expression by the T cells, and Fas on infected epithelial cells or lymphocytes. Animals with P(-/-) T cells and a Fas(-/-) lung failed to limit the shedding of infectious virus, regardless of whether CD4 T cells were present. In addition, we noted that having P(-/-) T cells in irradiated Fas(+/+) hosts caused a lethal disease that was not apparent in the non-chimeric (unirradiated) P(-/-) (Fas(+/+)) mice. In another set of chimeric mice, P(-/-) T cells were able to limit persistent infection of B cells that expressed Fas, but not B cells that were Fas-deficient. These studies demonstrate that some degree of cytotoxicity via either perforin or Fas is essential for CD8(+) T cells to control this DNA virus.