Carly Lodewyks

University of Manitoba, Winnipeg, Manitoba, Canada

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Publications (4)8.66 Total impact

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    ABSTRACT: Peptide hydrolysate supplements in mammalian cell cultures provide enhanced growth and productivity. The objective of this study was to compare the bioactivity of ten different commercially available hydrolysates from plant, microbial and animal sources. The peptide hydrolysates were tested as supplements to cultures of Chinese hamster ovary (CHO) cells that produce human beta interferon (β-IFN). A soy hydrolysate was shown to support high cell growth but not protein productivity compared to an animal component hydrolysate (Primatone RL). On the other hand a yeast hydrolysate showed lower cell growth but comparable productivity of the recombinant protein. Glycosylation analysis showed that the glycan profile of β-IFN produced in yeast hydrolysate supplemented cultures was equivalent to that from Primatone RL-supplemented cultures. Fractionation of the yeast hydrolysate and Primatone RL produced a similar protein-assayed pattern except for one extra peak at around 1 kDa in the Primatone RL profile. A fraction taken at a molecular weight range of 1.5 to 1.7 kDa showed the highest growth promoting activity in both samples. However, four other fractions in yeast hydrolysate and two in Primatone RL at lower molecular weights showed some growth promoting activity. In conclusion, the yeast hydrolysates provided a good alternative to the animal sourced Primatone RL for high productivity of β-IFN from CHO cells with equivalent glycosylation. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 2014.
    Biotechnology Progress 05/2014; · 1.85 Impact Factor
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    ABSTRACT: Aim:  Activation of adrenergic receptors (AR) has been reported to enhance the growth and invasion of various malignancies. The effects of AR agonists on malignant hepatocyte proliferation and migration have yet to be determined. Methods:  PLC/PRF/5 (PLC) and Huh-7 cells were exposed to a wide range of concentrations of the AR agonists noradrenaline (NA) and isoprenaline. Cell proliferation, migration, intracellular cyclic adenosine monophosphate (cAMP), protein kinase A (PKA) and C (PKC), matrix metalloproteinases (MMP)-2, -3, -7 and -9, and α(1) -, β(1) - and β(2) -AR expression were documented in both cell lines. Results:  Cell proliferative activity was unaltered following exposure to physiological and stress-related concentrations of AR agonists but migration was accelerated, an effect that was inhibited by the nonselective β-AR antagonist labetalol. cAMP, PKA, PKC or MMP expression remained unchanged. Although α(1) - and β(1) -AR expressions were abundant, β(2) -AR expression was limited in both cell lines. Conclusion:  Unlike other malignancies studied to date, in this study, the proliferative activity of malignant hepatocytes was not increased by exposure to AR agonists, a finding that could be explained by downregulation of β(2) -AR expression. The increase in malignant hepatocyte migration observed remains unexplained but does not appear to involve adenyl cyclase or MMP signaling pathways.
    Hepatology Research 10/2011; 41(10):1000-8. · 2.07 Impact Factor
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    ABSTRACT: There are conflicting data regarding whether activation of γ-aminobutyric acid-B (GABA-B) receptors results in inhibition of tumor growth and invasion. The objectives of this study were to document the effects of the GABA-B receptor agonist baclofen on malignant hepatocyte proliferation and migration. We also sought to determine whether any effects on cell migration were mediated by changes in cyclic adenosine monophosphate (cAMP) signaling or matrix metalloproteinase (MMP) expression. Finally, GABA-B(1) and -B(2) receptor expression was documented in 2 malignant hepatocyte cell lines (PLC/PRF/5 and Huh-7) and 12 sets of human hepatocellular carcinoma and adjacent nontumor tissues. Cell proliferative activity was documented by WST-1 absorbance, migration by wound healing assays, cAMP levels by enzyme-linked immunoassay (ELISA), MMP by immunohistochemistry and ELISA, and GABA-B receptor expression by flow cytometry and reverse transcriptase - polymerase chain reaction. Although baclofen had no effect on cell proliferation, wound healing was delayed, an effect that was reversed by the GABA-B receptor antagonist CGP. cAMP levels were decreased in Huh-7 but not PLC cells exposed to baclofen. MMP expression remained unaltered in both cell lines. Finally, GABA-B(1) receptor expression was present and consistently expressed, but GABA-B(2) expression was limited and varied with the number of cell passages and (or) duration of culture. In conclusion, activation of GABA-B receptors has no effect on malignant hepatocyte proliferation but does decrease cell migration. This inhibitory effect may involve cAMP signaling but not MMP expression. GABA-B(2) receptor expression is limited and variable, which may help to explain discrepancies with previously published results.
    Canadian Journal of Physiology and Pharmacology 06/2011; 89(6):393-400. · 1.56 Impact Factor
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    ABSTRACT: Recombinant human interferon-beta (β-IFN), used in the therapeutic treatment of multiple sclerosis (MS), can be produced on a large-scale from genetically engineered Chinese hamster ovary (CHO) cells. However, its hydrophobicity causes non-reversible, molecular aggregation in culture. The parameters affecting aggregation were determined to be concentration, culture residence time, temperature and glycosylation. Although the protein can be produced in Escherichia coli in a non-glycosylated form, the addition of glycans confers a reduced rate of aggregation as well as a 10-fold higher bioactivity. We report on the application of a low temperature perfusion culture designed to control the parameters that cause aggregation. In this three-phase culture system there is a transition to a low temperature (32°C) in a batch mode prior to implementing perfusion at 1 volume/day using an acoustic cell separator. Perfusion at the low temperature resulted in a 3.5-fold increase in specific productivity and a 7-fold increase in volumetric productivity compared to the batch culture at 37°C. The percentage aggregation of β-IFN was reduced from a maximum of 43% in batch culture to a minimum of 5% toward the end of the perfusion phase. The glycosylation profile of all samples showed predominantly sialylated biantennary fucosylated structures. The extent of sialylation, which is important for bioactivity, was enhanced significantly in the perfusion culture, compared to the batch culture.
    Journal of Biotechnology 10/2010; 150(4):509-18. · 3.18 Impact Factor