Manufacture of measles viruses.

Measles viruses have shown potent oncolytic activity as a therapeutic against a variety of human cancers in animal models and are currently being tested in clinical trials in patients. In contrast to using measles virus as a vaccine, oncolytic activity depends on high concentrations of infectious virus. For use in humans, the high-titer measles virus preparations must also be purified to remove significant levels of cellular proteins and nucleic acid resulting from the cytolytic products of measles virus replication and release. Pleomorphic measles virus must be treated as >1-µm particles that are extremely shear sensitive to maximize recoveries and retain infectivity. Therefore, to maximize the recovery of sterile, high titer infectious measles viruses, the entire production and purification process must be done using gentle conditions and aseptic processing. Here we describe a procedure applicable to the production of small (a few liters) to large (50-60 L) batches of measles virus amplified in Vero cells adapted to serum-free growth. Cell culture supernatant containing the measles virus is clarified by filtration to remove intact Vero cells and other debris, and then treated with Benzonase(®) in the presence of magnesium chloride to digest contaminating nucleic acid. The measles virus in the treated cell culture supernatant is then concentrated and purified using tangential flow filtration (TFF) and diafiltration. The concentrated and diafiltered measles virus is passed through a final clarifying filter prior to final vialing and storage at <-65°C. An infectivity assay to quantify infectious measles virus concentration based on the TCID(50) method is also described. This procedure can be readily adapted to the production and purification of measles viruses using good manufacturing practices (GMP).

Lixazinone stimulates mitogenesis of Madin-Darby canine kidney cells.

Polycystic kidney diseases (PKD) are characterized by excessive proliferation of renal tubular epithelial cells, development of fluid-filled cysts, and progressive renal insufficiency. cAMP inhibits proliferation of normal renal tubular epithelial cells but stimulates proliferation of renal tubular epithelial cells derived from patients with PKD. Madin-Darby canine kidney (MDCK) epithelial cells, which are widely used as an in vitro model of cystogenesis, also proliferate in response to cAMP. Intracellular cAMP levels are tightly regulated by phosphodiesterases (PDE). Isoform-specific PDE inhibitors have been developed as therapeutic agents to regulate signaling pathways directed by cAMP. In other renal cell types, we have previously demonstrated that cAMP is hydrolyzed by PDE3 and PDE4, but only PDE3 inhibitors suppress proliferation by inhibiting Raf-1 activity (Cheng J, Thompson MA, Walker HJ, Gray CE, Diaz Encarnacion MM, Warner GM, Grande JP. Am J Physiol Renal Physiol 287:F940-F953, 2004.) A potential role for PDE isoform(s) in cAMP-mediated proliferation of MDCK cells has not previously been established. Similar to what we have previously found in several other renal cell types, cAMP hydrolysis in MDCK cells is directed primarily by PDE4 (85% of total activity) and PDE3 (15% of total activity). PDE4 inhibitors are more effective than PDE3 inhibitors in increasing intracellular cAMP levels in MDCK cells. However, only PDE3 inhibitors, and not PDE4 inhibitors, stimulate mitogenesis of MDCK cells. PDE3 but not PDE4 inhibitors activate B-Raf but not Raf-1, as assessed by an in vitro kinase assay. PDE3 but not PDE4 inhibitors activate the ERK pathway and activate cyclins D and E, as assessed by histone H1 kinase assay. We conclude that mitogenesis of MDCK cells is regulated by a functionally compartmentalized intracellular cAMP pool directed by PDE3. Pharmacologic agents that stimulate PDE3 activity may provide the basis for new therapies directed toward reducing cystogenesis in patients with PKD.

Differential regulation of mesangial cell mitogenesis by cAMP phosphodiesterase isozymes 3 and 4.

Mesangial cell (MC) mitogenesis is regulated through "negative cross talk" between cAMP-PKA and ERK signaling. Although it is widely accepted that cAMP inhibits mitogenesis through PKA-mediated phosphorylation of Raf-1, recent studies have indicated that cAMP-mediated inhibition of mitogenesis may occur independently of Raf-1 phosphorylation or without inhibiting ERK activity. We previously showed that MCs possess functionally compartmentalized intracellular pools of cAMP that are differentially regulated by cAMP phosphodiesterases (PDE); an intracellular pool directed by PDE3 but not by PDE4 suppresses mitogenesis. We therefore sought to determine whether there was a differential effect of PDE3 vs. PDE4 inhibitors on the Ras-Raf-MEK-ERK pathway in cultured MC. Although PDE3 and PDE4 inhibitors activated PKA and modestly elevated cAMP levels to a similar extent, only PDE3 inhibitors suppressed MC mitogenesis (-57%) and suppressed Raf-1 kinase and ERK activity (-33 and -68%, respectively). Both PDE3 and PDE4 inhibitors suppressed B-Raf kinase activity. PDE3 inhibitors increased phosphorylation of Raf-1 on serine 43 and serine 259 and decreased phosphorylation on serine 338; PDE4 inhibitors were without effect. Overexpression of a constitutively active MEK-1 construct reversed the antiproliferative effect of PDE3 inhibitors. PDE3 inhibitors also reduced cyclin A levels (-27%), cyclin D and cyclin E kinase activity (-30 and -50%, respectively), and induced expression of the cell cycle inhibitor p21 (+90%). We conclude that the antiproliferative effects of PDE3 inhibitors are mechanistically related to inhibition of the Ras-Raf-MEK-ERK pathway. Additional cell cycle targets of PDE3 inhibitors include cyclin A, cyclin D, cyclin E, and p21.

Differential effects of low-dose docosahexaenoic acid and eicosapentaenoic acid on the regulation of mitogenic signaling pathways in mesangial cells.

Although dietary fish oil supplementation has been used to prevent the progression of kidney disease in patients with IgA nephropathy, relatively few studies provide a mechanistic rationale for its use. Using an antithymocyte (ATS) model of mesangial proliferative glomerulonephritis, we recently demonstrated that fish oil inhibits mesangial cell (MC) activation and proliferation, reduces proteinuria, and decreases histologic evidence of glomerular damage. We therefore sought to define potential mechanisms underlying the antiproliferative effect of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), the predominant omega-3 polyunsaturated fatty acids found in fish oil, in cultured MC. DHA and EPA were administered to MC as bovine serum albumin fatty-acid complexes. Low-dose (10-50 micromol/L) DHA, but not EPA, inhibited basal and epidermal growth factor (EGF)-stimulated [(3)H]-thymidine incorporation in MCs. At higher doses (100 micromol/L), EPA and DHA were equally effective in suppressing basal and EGF-stimulated MC mitogenesis. Low-dose DHA, but not EPA, decreased ERK activation by 30% (P <.01), as assessed with Western-blot analysis using phosphospecific antibodies. JNK activity was increased by low-dose DHA but not by EPA. p38 activity was not significantly altered by DHA or EPA. Cyclin E activity, as assessed with a histone H1 kinase assay, was inhibited by low-dose DHA but not by EPA. DHA increased expression of the cell cycle inhibitor p21 but not p27; EPA had no effect on p21 or p27. We propose that the differential effect of low-dose DHA vs EPA in suppressing MC mitogenesis is related to down-regulation of ERK and cyclin E activity and to induction of p21.

TGF-beta1 is an autocrine mediator of renal tubular epithelial cell growth and collagen IV production.

Recent studies in cultured cells have provided evidence that a variety of pathobiologic stimuli, including high glucose, angiotensin II, and thromboxane A(2), trigger a signaling pathway leading to autocrine induction of TGF-beta1. TGF-beta1 production through this pathway may profoundly affect cell growth, matrix synthesis, and response to injury. This study examines the role of autocrine versus exogenously added TGF-beta1 in cellular proliferation and collagen IV production, critical targets of TGF-beta1 signaling, using renal cells derived from TGF-beta1 knockout (KO) animals or wild-type (WT) controls. Growth of WT and KO cells was assessed by cell counting and [(3)H]thymidine uptake. Basal and TGF-beta1-stimulated collagen production was assessed by Northern and Western blotting; transcriptional activity of the alpha1(IV) collagen gene was assessed by transient transfection analysis. KO cells grew at a faster rate than WT cells carefully matched for plating density and passage number. This increased growth rate was paralleled by increases in [(3)H]thymidine uptake. KO cells expressed lower levels of the cell cycle inhibitors p21 and p27 than WT cells. KO cells failed to express TGF-beta1, as expected. Basal TGF-beta3 mRNA levels were higher in KO cells than in WT cells. WT cells expressed higher basal levels of TGF-beta2 mRNA than KO cells. Basal alpha1(IV) and alpha2(IV) collagen mRNA and protein expression were significantly lower in KO cells than WT cells. Administration of exogenous TGF-beta1 induced collagen IV production in both KO and WT cells. Although basal transcriptional activity of an alpha1(IV) collagen-CAT construct was lower in KO cells than WT cells, administration of exogenous TGF-beta1 was associated with significant increases in transcriptional activity of this construct in both KO and WT cells. These studies provide evidence that autocrine production of TGF-beta1 may play a critical role in regulation of growth and basal collagen IV production by renal tubular epithelial cells.