In vitro Susceptibility of Human Cell Lines Infection by Bovine Leukemia Virus.

Evidence of the presence of bovine leukemia virus (BLV) in human beings and its association with breast cancer has been published in the literature, proposing it as a zoonotic infection. However, not enough evidence exists about transmission pathways nor biological mechanisms in human beings. This study was aimed at gathering experimental evidence about susceptibility of human cell lines to BLV infection. Malignant and non-malignant human cell lines were co-cultured with BLV-infected FLK cells using a cell-to-cell model of infection. Infected human cell lines were harvested and cultured for 3 to 6 months to determine stability of infection. BLV detection was performed through liquid-phase PCR and visualized through in situ PCR. Seven out of nine cell lines were susceptible to BLV infection as determined by at least one positive liquid-phase PCR result in the 3-month culture period. iSLK and MCF7 cell lines were able to produce a stable infection throughout the 3-month period, with both cytoplasmic and/or nuclear BLV-DNA visualized by IS-PCR. Our results support experimental evidence of BLV infection in humans by demonstrating the susceptibility of human cells to BLV infection, supporting the hypothesis of a natural transmission from cattle to humans.

Recombinant Expression of Complex Proteins in Human Cell Lines.

Coagulation factors, as factor VII, VIII, and IX, are complex proteins which are very difficult to express. Blood coagulation factor IX is a vitamin K-dependent protein, and it has become a valuable biopharmaceutical in the treatment of hemophilia B. Here, we describe the techniques used to generate human cell lines producing human recombinant factor IX, as an example of complex protein, as well as in vitro characterization of this coagulation factor.To produce the FIX human adherent 293T SK-Hep-1 cells were used and stably modified by a lentiviral vector carrying the hFIX and the eGFP genes. The eGFP was employed as a reporter protein.

Exploiting Zebrafish Xenografts for Testing the in vivo Antitumorigenic Activity of Microcin E492 Against Human Colorectal Cancer Cells.

One of the approaches to address cancer treatment is to develop new drugs not only to obtain compounds with less side effects, but also to have a broader set of alternatives to tackle the resistant forms of this pathology. In this regard, growing evidence supports the use of bacteria-derived peptides such as bacteriocins, which have emerged as promising anti-cancer molecules. In addition to test the activity of these molecules on cancer cells in culture, their in vivo antitumorigenic properties must be validated in animal models. Although the standard approach for such assays employs experiments in nude mice, at the initial stages of testing, the use of high-throughput animal models would permit rapid proof-of-concept experiments, screening a high number of compounds, and thus increasing the possibilities of finding new anti-cancer molecules. A validated and promising alternative animal model are zebrafish larvae harboring xenografts of human cancer cells. Here, we addressed the anti-cancer properties of the antibacterial peptide microcin E492 (MccE492), a bacteriocin produced by Klebsiella pneumoniae, showing that this peptide has a marked cytotoxic effect on human colorectal cancer cells in vitro. Furthermore, we developed a zebrafish xenograft model using these cells to test the antitumor effect of MccE492 in vivo, demonstrating that intratumor injection of this peptide significantly reduced the tumor cell mass. Our results provide, for the first time, evidence of the in vivo antitumoral properties of a bacteriocin tested in an animal model. This evidence strongly supports the potential of this bacteriocin for the development of novel anti-cancer therapies.

Strategies to Suspension Serum-Free Adaptation of Mammalian Cell Lines for Recombinant Glycoprotein Production.

Serum-free suspension cultures are preferably required for recombinant protein production due to its readiness in upstream/downstream processing and scale-up, therefore increasing process productivity and competitiveness. This type of culture replaces traditional cell culturing as the presence of animal-derived components may introduce lot-a-lot variability and adventitious pathogens to the process. However, adapting cells to serum-free conditions is challenging, time-consuming, and cell line and medium dependent. In this chapter, we present different approaches that can be used to adapt mammalian cell lines from an anchorage-dependent serum supplemented culture to a suspension serum-free culture.

Human Cells as Platform to Produce Gamma-Carboxylated Proteins.

The gamma-carboxylated proteins belong to a family of proteins that depend on vitamin K for normal biosynthesis. The major representative gamma-carboxylated proteins are the coagulation system proteins, for example, factor VII, factor IX, factor X, prothrombin, and proteins C, S, and Z. These molecules have harbored posttranslational modifications, such as glycosylation and gamma-carboxylation, and for this reason they need to be produced in mammalian cell lines. Human cells lines have emerged as the most promising alternative to the production of gamma-carboxylated proteins. In this chapter, the methods to generate human cells as a platform to produce gamma-carboxylated proteins, for example the coagulation factors VII and IX, are presented. From the cell line modification up to the vitamin K adaptation of the produced cells is described in the protocols presented in this chapter.

Production of recombinant coagulation factors: Are humans the best host cells?

The main treatment option for Hemophilia A/B patients involves the administration of recombinant coagulation factors on-demand or in a prophylactic approach. Despite the safety and efficacy of this replacement therapy, the development of antibodies against the coagulation factor infused, which neutralize the procoagulant activity, is a severe complication. The production of recombinant coagulation factors in human cell lines is an efficient approach to avoid such complication. Human cell lines can produce recombinant proteins with post translation modifications more similar to their natural counterpart, reducing potential immunogenic reactions. This review provides a brief overview of the most important characteristics of recombinant FVIII and FIX products available on the market and the improvements that have recently been achieved by the production using human cell lines.

Identification of reference genes for quantitative real-time PCR studies in human cell lines under copper and zinc exposure.

Accurate quantification depends on normalization of the measured gene expression data. In particular, gene expression studies with exposure to metals are challenging due their toxicity and redox-active properties. Here, we assessed the stability of potential reference genes in three cell lines commonly used to study metal cell metabolism: Caco-2 (colon), HepG2 (liver) and THP-1 (peripheral blood) under copper (Cu) or zinc (Zn) exposure. We used combined statistical tools to identify the best reference genes from a set of eleven candidates, which included traditional "housekeeping" genes such as GAPDH and B-ACTIN, in cell lines exposed to high and low, Zn and Cu concentrations. The expression stabilities of ATP5B (ATP synthase) and CYC1 (subunits of the cytochrome) were the highest considering the effect of Zn and Cu treatments whereas SDHA (succinate dehydrogenase) was found to be the most unstable gene. Even though the transcriptional effect of Zn and Cu is very different in term of redox properties, the same best reference genes were identified when Zn or Cu treatments were analyzed together. Our results indicate that ATP5B/CYC1 are the best candidates for reference genes after metal exposure, which can be used as a suitable starting point to evaluate gene expression with other metals or in different cell types in human models.

Antiproliferative effect of extracts and pyranonaphthoquinones obtained from Cipura paludosa bulbs.

CONTEXT: Cipura paludosa Aubl. (Iridaceae) is widely used in folk medicine to treat several ailments. Experimental studies have confirmed its anti-inflammatory, antinociceptive, and neuroprotective effects. OBJECTIVE: This study evaluates the possible antiproliferative potential of the crude methanol extract and three isolated compounds from the bulbs of C. paludosa. MATERIALS AND METHODS: Phytochemical analysis was carried out by conventional chromatographic techniques, and the resulting compounds were identified by NMR (1)H and (13)C. The antiproliferative activity was analysed using the sulforhodamine B assay. RESULTS: Crude methanol extract of C. paludosa bulbs showed GI50 values of between 1.6 and 30.8 µg/mL. The naphthoquinone derivatives (eleutherine, isoeleutherine, and eleutherol) isolated from the bulbs of C. paludosa exhibited promising cytotoxicity against several human tumour cell lines, especially the two main compounds, eleutherine and isoeleutherine, against glioma and breast cancer cell lines, with TGI values of between 2.6 and 13.8 µg/mL. CONCLUSION: Cipura paludosa bulbs produce active principles with relevant antiproliferative potential, such as naphthoquinone derivatives, identified as eleutherine, isoeleutherine, and eleutherol. This is the first report indicating C. paludosa with antiproliferative potential.

Antiproliferative activity of methanol extracts of four species of Croton on different human cell lines

Several species of Croton have been described with biological activities, mainly due to diterpenes, alkaloids and/or other secondary metabolites. These activities account for the traditional use of Croton species to treat certain diseases in South America, Asia and Western Africa. The crude methanol extracts obtained from leaves and steam bark of Croton dichrous Müll. Arg., C. erythroxyloides Baill., C. myrianthus Müll. Arg. and C. splendidus Mart. ex Colla were tested for antiproliferative activity against ten human cancer cell lines. Chemical analyses of all extracts were carried out by GC/MS and HPLC/MS/MS. The leaf extract obtained from C. erythroxyloides showed potent activity against PC-3 (prostate) and OVCAR-3 (ovary) cell lines. Lupeol is suggested to be involved in such activity. Tiliroside, an acyl-glycosilated flavonoid ubiquitous in all tested extracts, seems to play an important role in the observed moderate activity of most extracts against the leukemia K562 cell lineage.

Diversity of marine gliding bacteria in Thailand and their cytotoxicity

Eighty-four marine gliding bacteria were isolated from specimens collected in the Gulf of Thailand and the Andaman Sea. All exhibited gliding motility and swarm colonies on cultivation plates and they were purified by subculturing and micromanipulator techniques. Their 16S rRNA genes were amplified by the polymerase chain reaction (PCR) technique. The phylogenetic analysis indicated that the represented isolates can be separated into six different clads (gr 1 - gr 6) within the Cytophaga-Flavobacterium-Bacteriodes (CFB) group. Group 1 formed a remote linear, with only 90 percent sequence similarity, from Flavobacteriaceae bacterium which indicated a potentially novel taxonomic group. Groups 2 and 3 were identified as the recently proposed Tenacibaculum mesophilum and Fulvivirga kasyanovii respectively. Groups 4, 5 and 6, consisting of the largest number of the members, were identified as Rapidithrix thailandica, Aureispira marina and Aureispira maritima respectively. The isolates were cultivated in four different cultivation media (Vy/2, RL 1, CY and SK) and the crude extracts were submitted to screen cytotoxicity using a sulphorodamine B (SRB) assay. The results from cytotoxic screening showed that groups 2, 4 and 6 were capable of producing the cytotoxic metabolites against selected human cell lines (breast adenocarcinoma (MCF-7), colon cancer (HT-29), cervical cancer (HeLa) and oral cancer (KB)). However, groups 1, 3 and 5 did not produce metabolites with cytotoxicity when cultivated in the same cultivation media as the previous groups. CY medium was the only cultivation medium which could yield the cytotoxic metabolites against MCF-7.