Characterization of a quail suspension cell line for production of a fusogenic oncolytic virus

The development of efficient processes for the production of oncolytic viruses (OV) plays a crucial role regarding the clinical success of virotherapy. Although many different OV platforms are currently under investigation, manufacturing of such viruses still mainly relies on static adherent cell cultures, which bear many challenges, particularly for fusogenic OVs. Availability of GMP-compliant continuous cell lines is limited, further complicating the development of commercially viable products. BHK21, AGE1. CR and HEK293 cells were previously identified as possible cell substrates for the recombinant vesicular stomatitis virus (rVSV)-based fusogenic OV, rVSV-NDV. Now, another promising cell substrate was identified, the CCX.E10 cell line, developed by Nuvonis Technologies. This suspension cell line is considered non-GMO as no foreign genes or viral sequences were used for its development. The CCX.E10 cells were thus thoroughly investigated as a potential candidate for OV production. Cell growth in the chemically defined medium in suspension resulted in concentrations up to 8.9 × 10⁶ cells/mL with a doubling time of 26.6 h in batch mode. Cultivation and production of rVSV-NDV, was demonstrated successfully for various cultivation systems (ambr15, shake flask, stirred tank reactor, and orbitally shaken bioreactor) at vessel scales ranging from 15 mL to 10 L. High infectious virus titers of up to 4.2 × 10⁸ TCID₅₀/mL were reached in orbitally shaken bioreactors and stirred tank reactors in batch mode, respectively. Our results suggest that CCX.E10 cells are a very promising option for industrial production of OVs, particularly for fusogenic VSV-based constructs.     

Study of the Biological Activity and Phenolic Content of the Ethanol Extract of Phyllogonium viride Brid. (Phyllogoniaceae,Bryophyta)

The present study aimed to examine the phenolic content and evaluate the antimicrobial and antioxidant potential of ethanol extracts from the moss species Phyllogonium viride Brid. on the pathogenic bacteria Salmonella enterica serovar enteritidis, Staphylococcus aureus, Listeria monocytogenes and Escherichia coli, and the pathogenic fungi Candida albicans and Cryptococcus neoformans. The antimicrobial activity was determined from Minimum Inhibitory Concentration (MIC) Minimum Bactericidal Concentration (MBC) and Minimum Fungicidal Concentration (MFC). Antioxidant activity was determined by the DPPH method. Folin-Denis reagent was used for the content of total phenolics and flavonoids and HPLC-DAD for identification of phenolic compounds. The results showed that bacteriostatic and bactericidal activities occurred at concentrations ranging from 9.76 μg/mL–78.13 μg/mL among all evaluated microorganisms. These values, considering the criteria used, suggest the P. viride extract as a potent antimicrobial. For antioxidant activity, P. viride extract was considered weak. Analysis of the phenolic content showed a wide range of compounds, with Kaempferol (0.41 mg/g) being the major compound, followed by t-cinnamic acid and caffeic acid (0.17 mg/g). Although P. viride is a species of moss not yet referenced in scientific publications of biotechnological interest, it has shown promising potential for further studies and possible application as an antimicrobial of natural origin.     

Irradiation of cells in tissue culture

Summary Cultures of human amnion were employed to check the hypothesis that cell strains with heteroploid chromosome counts regularly produce giant cells within 12 days following treatment with 2000 r and 4000 r of gamma irradiation from a cobalt source, while this response has not been obtained from primary cultures whose cells were presumed to be diploid.The giant cell reaction not only was obtained from two transfer passage lines of a well-established amnion strain developed at Berkeley (No A 185-21C-26 and No A 185-21C-45) but was also found for a 20-day second passage culture of amnion. Since this line has continued to reproduce at a rapid rate, it is presumed to have assumed the features of a typical strain within the period of observation. This impression was reinforced by the finding that the chromosome number for 32 cells fixed on the 35th day had a modal value of 67.In contrast, both untrypsinized and trypsinized spindle cells in primary cultures as well as unaltered epithelial elements which had not been subcultured gave no evidence of giant cell formation 12 days after exposure to 2000 r and 4000 r from a Cobalt⁶⁰ source.These data lend evidence that giant cell formation is related to the chromosomal constitution of the irradiated elements.This research was supported by funds provided under Contract AF 18(600)-1263 with the School of Aviation Medicine, USAF, Randolph Air Force Base, Texas.Fellow of the Instituto de Alta Cultura and the Fundacão Calouste Gulbenkian of Lisbon, Portugal.Tobacco Industry Research Committee Fellow.     

Human papilloma virus E6/E7 genes can expand the lifespan of human corneal fibroblasts

Summary Human corneal fibroblasts were infected with a retroviral delivery vector containing the E6 and E7 genes from human Papilloma virus type 16 in order to produce cell lines that have an expanded lifespan in culture. Morphologically, some of the trasfected corneal fibroblast lines appeared to have the normal spindle-shape morphology of diploid fibroblasts, whereas other lines appeared to have a more elongated morphology. All the cell lines were anchorage-dependent. Cells that had a normal morphology grew at a rate similar to normal diploid human corneal fibroblasts and had a population doubling time of 48 h. All E6/E7 expressing cell lines, regardless of morphology, produce types I, III, and V collagen, at levels similar to those observed in the parent corneal diploid fibroblast. These corneal fibroblast lines will be a usefulin vitro system to study collagen expression and fibril formation, as well as normal stroma development. These results also demonstrate that the use of E6/E7 genes to expand a cell’s lifespan can be a powerful tool because it does not appear to alter either the growth rate of the cell or collagen expression.     

Dihydrochalcones, coumarins and alkaloids from Metrodorea nigra

Two novel dihydrochalcones, 2′,3,4′,6′-tetrahydroxy-4-methoxy-3′,5-di-(3,3-dimethylallyl)-dihydrochalcone and 2′,.3,6′-trihydroxy-4-methoxy-5-(3,3-dimethylallyl)-3′,4′-(2″,2″-dimethyldihydropyran)-dihydrochalcone, have been isolated from fresh fruits of Metrodorea nigra. Stems and leaves showed a similar composition and we have isolated common steroids, simple coumarins, several furocoumarins, furoquinoline alkaloids and a furofuran lignan. From stems, we have also isolated the pentacyclic 6-C-monoterpenyl-5,7-dioxycoumarin, deoxybruceol. Structures of the isolated compounds were elucidated on the basis of spectral data.     

Extracellular polysaccharide of Nostoc commune (Cyanobacteria) inhibits fusion of membrane vesicles during desiccation

Cells of the cyanobacterium Nostoc commune secrete a complex, high molecular weight, extracellular polysaccharide (EPS) which accumulates to more than 60% of the dry weight of colonies. The EPS was purified from the clonal isolate N. commune DRH1. The midpoint of the membrane phase transition (T_m) of desiccated cells of N. commune CHEN was low (T_{m dry} = 8 °C) and was comparable to the T_m of rehydrated cells((T_m)_H20 = 6 °C). The EPS was not responsible for the depression of T_m. However, the EPS, at low concentrations, inhibited specifically the fusion of phosphatidylcholine membrane vesicles when they were dried in vitro at0% relative humidity (−400 MPa). Low concentrations of a trehalose:sucrose mixture, in a molar ratio which corresponded with that present in cells in vivo, together with small amounts of the EPS, were efficient in preventing leakage of carboxyfloroscein (CF) from membrane vesicles. Freeze-fracture electron microscopy resolved complex changes in the structure of the EPS and the outer membrane in response to rehydration of desiccated cells. The capacity of the EPS to prevent membrane fusion, the maintenance of a low T_{m dry} in desiccated cells, and the changes in rheological properties of the EPS in response to water availability, constitute what are likely important mechanisms for desiccation tolerance in this cyanobacterium. This revised version was published online in August 2006 with corrections to the Cover Date.     

Polyamine derivatives as inhibitors of trypanothione reductase and assessment of their trypanocidal activities

Trypanothione reductase (TR) occurs exclusively in trypanosomes and leishmania, which are the etiological agents of many diseases. TR plays a vital role in the antioxidant defenses of these parasites and inhibitors of TR have potential as antitrypanosomal agents. We describe the syntheses of several spermine and spermidine derivatives and the inhibiting effects of these compounds on T. cruzi TR. All of the inhibiting compounds displayed competitive inhibition of TR-mediated reduction of trypanothione disulfide. The three most effective compounds studied were N⁴,N⁸-bis(3-phenylpropyl)spermine (12), N⁴,N⁸-bis(2-naphthylmethyl)spermine (14), and N¹,N⁸-bis(2-naphthylmethyl)spermidine (21), with K_i values of 3.5, 5.5 and 9.5 μM, respectively. Compounds 12, 14, and 21 were found to be potent trypanocides in vitro with IC₅₀ values ranging from 0.19 to 0.83 μM against four T. brucei ssp. strains. However, these compounds did not prolong the lives of mice infected with trypanosomes. This work indicates that certain polyamine derivatives which target a unique pathway in Trypanosomatidae have potential as antitrypanosomal agents.