Continuous Cell Lines Research Articles

Characterization and virus susceptibility of a continuous cell line derived from the brain of Aequidens rivulatus (Günther).

Cell cultures derived from the brain tissues of Aequidens rivulatus (Günther) have been characterized previously. In this study, a continuous cell line ARB8 was further established, and its growth characteristics, transcription and susceptibility to fish viruses-including chum salmon reovirus (CSV), marbled eel infectious pancreative necrosis virus (MEIPNV), grouper nervous necrosis virus (GNNV), giant seaperch iridovirus (GSIV), red seabream iridovirus (RSIV), koi herpesvirus (KHV), herpesvirus anguilla (HVA) and marbled eel polyoma-like virus (MEPyV)-were examined. ARB8 cells that showed epithelioid morphology and were passaged >80 times grew well at temperatures ranging from 25°C to 30°C in L-15 medium containing 5%-15% foetal bovine serum. The cells constitutively transcribed connexion 43, glutamine synthetase, nestin and nkx6-2, which are markers for neural progenitor cells. The cells were highly susceptible to CSV, MEIPNV, GSIV and RSIV and showed the typical cytopathic effect (CPE). However, the cells were resistant to GNNV, KHV, HVA and MEPyV because no significant CPE was noted after infection. Optimal temperatures for virus production ranged from 25°C to 30°C. The results revealed that the neural progenitor cell line ARB8 can potentially serve as a useful tool for investigating fishvirusesand isolating newvirusesin ornamental cichlid fishes.

Assessment of titanium dioxide nanoparticle toxicity in the rainbow trout (Onchorynchus mykiss) liver and gill cell lines RTL-W1 and RTgill-W1 under particular consideration of nanoparticle stability and interference with fluorometric assays

The increasing release of titanium dioxide (TiO2) nanoparticles (NPs) into the environment calls for a thorough assessment of their hazard to aquatic organisms. In vitro-assays based on continuous fish cell lines bear a high potential for determining the relative cytotoxic potency and mode of toxic action of environmental contaminants. Their application for testing of manufactured nanomaterials is however concomitant with technical challenges. The objective of this study was to determine the acute cytotoxicity of TiO2 NP in the rainbow trout (Onchorynchus mykiss) liver and gill cell lines RTL-W1 and RTgill-W1 under special consideration of so far widely ignored issues arising from poor NP stability and NP-dependent interference with fluorometric assays. We validated a protocol for the preparation of serum albumin-stabilized TiO2 NP dispersions in two growth media, which are frequently used in the culture of fish cells, Leibovitz-15 (L-15) and L-15/ex. The TiO2 NPs dispersions displayed high colloidal stability with their size distribution remaining unchanged over a concentration range that spans two orders of magnitude (1–100 μg/ml) and an incubation period of 72 h. The TiO2 NPs dispersions caused little cytotoxicity upon 24 h and 72 h exposure, but NP agglomerates/aggregates were found inside intracellular vesicles in both gill and liver cells. Furthermore we demonstrated that TiO2 NPs, which remained adsorbed to the plasma membrane and/or were internalized by the cell interfere with fluorometric cell viability assays by attenuating part of the incident and emitted light resulting in effect overestimation. We propose modifications in the original assay protocol that may allow quantifying and mathematically correcting for the level of interference at different exposure concentrations.

Controversial truth: Human pancreatic cancer cell line homes cancer stem cells

Event Abstract Back to Event Controversial truth: human pancreatic cancer cell line homes cancer stem cells Yuan Han Teh1, Rajesh Ramasamy2 and Johnson Stanslas1* 1 Department of Medicine, Faculty of Medicine and Health Sciences, University Putra Malaysia, Malaysia 2 Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Malaysia Background Pancreatic cancer stem cells (pCSCs) are the culprit of pancreatic cancer recurrence. Disputable presence of pCSCs in continuous cell line has led to an extensive application of patient-derived pCSC culture for in vitro evaluation of anti-pCSC agents. Nevertheless, the procurement of patients’ tumour biopsies can be challenging to some researchers. Here, we investigated the presence of CSCs in pancreatic cancer cell lines (PANC-1, MIA PaCa-2, Capan-2, and BxPC-3). Methods Dose-response effect of gemcitabine and vismodegib on pancreatic cancer cell lines was evaluated by MTT cell viability assay. Immunophenotyping was achieved via flow cytometry. Limiting dilution spheroid formation assay and Extreme Limiting Dilution Analysis (ELDA) software determined in vitro tumourigenicity of Capan-2 cells. Tumourspheres were treated with gemcitabine and vismodegib, followed by counting intact tumourspheres. Results Capan-2 was the most resistant cell line to gemcitabine (IC50 > 100 µM). This profound chemoresistance coincided with the presence of a cell subpopulation (3.8%) that co-expressed pCSC surface markers (CD44, CD24, and CD133), which was undetectable in other cell lines. ELDA software estimated that 1.45 − 4.15% of Capan-2 cells were tumourigenic. These findings collectively imply that CD44+CD24+CD133+ subpopulation is potentially pCSCs, which survive chemotherapy and initiate recurrence. Capan-2 tumourspheres demonstrated molecular features of pCSCs as reported previously. GLI1 expression was down-regulated in these tumourspheres, suggesting an independence of canonical Hedgehog signaling. This finding explains an unexpectedly moderate response of CD44+CD24+CD133+ cells (p=0.738) and tumourspheres (IC50>20µM) to vismodegib treatment that targets canonical Hedgehog signaling. The integrity of tumourspheres was remarkably irresponsive to gemcitabine (IC50>100µM), which is in line with an enrichment of CD44+CD24+CD133+ subpopulation (p=0.002). Conclusion Capan-2 human pancreatic cancer cell line harbours CD44+CD24+CD133+ subpopulation that behaves like pCSCs, thus it is an attractive substitute for patients’ tumour biopsies for anti-pCSC drug discovery. Keywords: pancreatic cancer stem cells, chemoresistance, Hedgehog signaling, Tumourigenicity, Pancreatic cancer cell lines Conference: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”, Putrajaya, Malaysia, 3 Dec - 5 Feb, 2019. Presentation Type: Oral Presentation Topic: Cancer Citation: Teh Y, Ramasamy R and Stanslas J (2019). Controversial truth: human pancreatic cancer cell line homes cancer stem cells. Front. Pharmacol. Conference Abstract: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”. doi: 10.3389/conf.fphar.2018.63.00061 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 22 Sep 2018; Published Online: 17 Jan 2019. * Correspondence: Prof. Johnson Stanslas, Department of Medicine, Faculty of Medicine and Health Sciences, University Putra Malaysia, Serdang, Malaysia, jstanslas@yahoo.co.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Yuan Han Teh Rajesh Ramasamy Johnson Stanslas Google Yuan Han Teh Rajesh Ramasamy Johnson Stanslas Google Scholar Yuan Han Teh Rajesh Ramasamy Johnson Stanslas PubMed Yuan Han Teh Rajesh Ramasamy Johnson Stanslas Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Isolation, characterization and sequence determination of Noda virus from sea bass (Dicentrarchus labraxL.) reared in freshwater and marine facilities in Greece

The present study aims το characterize Noda virus isolates from freshwater and marine fish, specifically from species Dicentrarchus labrax (sea bass). Previous works reported the detection of Noda virus only by using PCR, in cultured Dicentrarchus labrax showing clinical symptoms. Here we describe the isolation of Noda virus from symptomatic sea bass cultured in sea and freshwater facilities in Greece. The virus was isolated in the continuous cell line SSN -1, exhibiting characteristic cytopathic effect, vacuolation and degeneration of the monolayer. In parallel, in all cerebral tissue samples isolated from infected individuals, a 255 bp viral fragment has been detected, using Reverse Transcription - Polymerase Chain Reaction (RT-PCR) and nested PCR. Comparison of the amplified sequence was detected in diseased fish in European farms and in other piscine species revealed a high nucleotide homology.

Establishment of cell line with NK/NKT phenotype from myeloid NK cell acute leukemia

Acute Myeloid Leukemia (AML) is the most common malignancy in adults with a 5-year survival rate of 27% of the total affected population. For effective treatment and new drug discovery, cell lines are considered as a very important tool. Here we report an establishment of a continuous human cell line AML-004 with a hypo-diploid chromosome 44 and presence of both NK/NKT phenotypes. The cell line was isolated from the blood sample of myeloid NK cell acute leukemia patients and extensively characterized by flow cytometery, morphology, and cytogentic analysis. Cytotoxicity by standard chemotherapeutic drugs was also examined. As characterized by Giemsa staining, the predominant cell type in the culture had high nuclear/cytoplasmic ratio. Cytogenetic analysis revealed high chromosome instability and structural abnormalities confirming the source of cell line from a patient with AML. The karyotype of the isolated cells did not alter up to around 40 passages. These AML-004 cells lacked specific markers for B and T lymphoid cells, but expressed surface receptors for lymphoid/NK cells. Cells also lacked the presence of early progenitors. The proliferation of the isolated cells was inversely proportional to the IL-2 concentration confirming presence of NK phenotype. AML-004 was resistant against standard chemotherapeutic drugs excluding cisplatin. Thus, AML-004 cells provide a continuous source of human cells for designing novel therapies for patients with T-lymphoblastic leukemia/lymphoma.

Biopolymer mediated nanoparticles synthesized from Adenia hondala for enhanced tamoxifen drug delivery in breast cancer cell line

Silver nanoparticles (AgNPs) are an important class of nanomaterials, which have used as antimicrobial and disinfectant agents due to their detrimental effect on target cells. In the present study it was explored to deliver a novel tamoxifen drug system that can be used in breast cancer treatment, based on chitosan coated silver nanoparticles on MCF-7 human breast cancer cells. AgNPs synthesized from Adenia hondala tuber extract were used to make the chitosan coated AgNPs (Ch-AgNPs), in which the drug tamoxifen was loaded on chitosan coated silver nanoparticles (Tam-Ch-AgNPs) to construct drug loaded nanoparticles as drug delivery system. The morphology and characteristics of the Ch-AgNPs were investigated by UV, FTIR, zeta potential and FESEM. Furthermore, the toxicity of AgNPs, Ch-AgNPs, Tam-Ch-AgNPs was evaluated through cell viability, lactate dehydrogenase leakage, reactive oxygen species generation, caspase-3, DNA laddering, and TUNEL assay in human breast cancer cells (MCF-7) and HBL-100 continuous cell line as a control. Treatment of cancer cells with various concentrations of AgNPs, Ch-AgNPs, Tam-Ch-AgNPs for 24 h revealed that Tam-Ch-AgNPs could inhibit cell viability and induce significant membrane leakage in a dose-dependent manner. Cells exposed to Tam-Ch-AgNPs showed increased reactive oxygen species and hydroxyl radical production when compared to AgNPs, Ch-AgNPs. Furthermore, the apoptotic effects of AgNPs, Ch-AgNPs, Tam-Ch-AgNPs were confirmed by activation of caspase-3 and DNA nuclear fragmentation. The present findings suggest that Tam-Ch-AgNPs could contribute to the development of a suitable anticancer drug delivery.

Establishment and characterization of a brain cell line from sea perch, Lateolabrax japonicus.

A continuous cell line, designated LJB, derived from the brain of sea perch (Lateolabrax japonicus) was established. LJB cells have been subcultured for more than 60 times in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum (FBS) since the initial primary culture. LJB cells exhibited maximum growth rate at 28°C in DMEM supplemented with 20% FBS. Cytogenetic analysis indicated that the modal chromosome number was 48, which was identical with the chromosome number of embryonic stem-like cells of sea perch. Comparison of the 18S ribosomal RNA gene sequences of LJB cells and sea perch confirmed that LJB cells originated from sea perch. After transfected with pEGFP-N3 plasmid, LJB cells showed a transfection efficiency of about 40% which was indicated by the percentage of cells expressing green fluorescence protein, indicating the potential application of LJB cells in gene expression studies. Cytopathic effect was clearly observed, and RNA-dependent RNA polymerase gene was also detected in LJB cells post red-spotted grouper nervous necrosis virus (RGNNV) infection. Furthermore, virus replication was confirmed by quantitative RT-PCR, virus titer, and transmission electron microscopy assay in RGNNV-infected LJB cells. The LJB cell line might be used as an ideal in vitro tool for analyzing and understanding the mechanisms of nervous necrosis virus-host interaction.

Standardization of DNA Residual Quantification Method of Vero Cell Rabies Vaccine for Human Use.

Objectives:Normalize the quantification of residual DNA from Vero cells in the rabies vaccine for use in human VAHV I, by quantitative PCR in real time and the design of primers that amplified, highly repetitive sequences of Cercopithecus aethiops and a constitutive gene according to sequences reported in the GenBank and quantifying the residual DNA in the vaccine VAHV I in three consecutive batches according to the standard set by the World Health Organization.Methods:A real time quantitative method based on SYBR Green chemistry has been applied for the quantification of residual DNA (resDNA) using highly repetitive DNA (Alu) and a housekeeping gene (B-actin) as target sequences.Results:The sensitivity achieved with this white sequence is within the reported limits and who are between 5 and 50 pg. For real time PCR optimization with Alu-p53, different concentrations of MgCl2 (0.5, 0.75, 1.0, 1.25 and 1.5 mm) in combination with three different concentrations of primers (75, 100 and 150nM) were used. pDNA in concentration of 1x107 copies / ul was used as template. Optimal concentrations were 1.25 mM MgCl2 and 100nM primers. To level of detection of 1.53 ng/ul was found for p53-Alu and Alu-Glob and 0.39 ng/ul for B-actin with gDNA curves.Conclusion:Quantification of resDNA of vaccine VAHV I with close-ups of B-actin was normalized. Reached a sensitivity of 30 pg of resDNA/dose VAHV I, with close-ups of B-actin. Found, in three consecutive batches, an amount less than 10 ng/dose, these results suggest that the production process ensures vaccine resDNA removal, meeting international requirements for biological products for use in humans that use continuous cell lines for production.

A reverse genetics system for avian coronavirus infectious bronchitis virus based on targeted RNA recombination

BackgroundAvian coronavirus infectious bronchitis virus (IBV) is a respiratory pathogen of chickens that causes severe economic losses in the poultry industry worldwide. Major advances in the study of the molecular biology of IBV have resulted from the development of reverse genetics systems for the highly attenuated, cell culture-adapted, IBV strain Beaudette. However, most IBV strains, amongst them virulent field isolates, can only be propagated in embryonated chicken eggs, and not in continuous cell lines.MethodsWe established a reverse genetics system for the IBV strain H52, based on targeted RNA recombination in a two-step process. First, a genomic and a chimeric synthetic, modified IBV RNA were co-transfected into non-susceptible cells to generate a recombinant chimeric murinized (m) IBV intermediate (mIBV). Herein, the genomic part coding for the spike glycoprotein ectodomain was replaced by that of the coronavirus mouse hepatitis virus (MHV), allowing for the selection and propagation of recombinant mIBV in murine cells. In the second step, mIBV was used as the recipient. To this end a recombination with synthetic RNA comprising the 3′-end of the IBV genome was performed by introducing the complete IBV spike gene, allowing for the rescue and selection of candidate recombinants in embryonated chicken eggs.ResultsTargeted RNA recombination allowed for the modification of the 3′-end of the IBV genome, encoding all structural and accessory genes. A wild-type recombinant IBV was constructed, containing several synonymous marker mutations. The in ovo growth kinetics and in vivo characteristics of the recombinant virus were similar to those of the parental IBV strain H52.ConclusionsTargeted RNA recombination allows for the generation of recombinant IBV strains that are not able to infect and propagate in continuous cell lines. The ability to introduce specific mutations holds promise for the development of rationally designed live-attenuated IBV vaccines and for studies into the biology of IBV in general.

Exendin-4 Exhibits Enhanced Anti-tumor Effects in Diabetic Mice

Type 2 diabetes (T2D) is associated with increased risk of cancers. In this connection, we previously demonstrated the promoting effect of diabetes on HPV-associated carcinogenesis using a xenograft model in db/db diabetic mice. The underlying mechanism of this observation might be partly contributed by dysregulated immune response in diabetes. In this study, we hypothesized that the impaired anti-tumor immune response in diabetic status could be modulated by exendin-4, a glucagon-like protein receptor agonist which exhibits anti-diabetic effects. We inoculated 10-week old db/db mice with 2 × 107 CUP-1 cells (Human Papilloma Virus (HPV)-16 E7 transfected continuous cell line) subcutaneously underneath the scruff, and treated mice with high (30 nmol/kg) or low (10 nmol/kg) dose of exendin-4 for 13 days. Compared with control groups, exendin-4 suppressed subcutaneous tumor growth in a dose-dependent manner, accompanied by increased interferon (IFN)-γ secreting CD8+ cytotoxic T lymphocyte (CTL)/Foxp3+ regulatory T cell (Treg) ratio as well as Th1 proinflammatory cytokines IFN-γ and IL-2. Collectively, these findings suggested an anti-tumor effect of exendin-4 in diabetic conditions, which might be resulted from direct immunomodulation.

Tenofovir Disoproxil Fumarate Is a New Substrate of ATP-Binding Cassette Subfamily C Member 11.

Tenofovir disoproxil fumarate (TDF), a nucleotide reverse transcriptase inhibitor, after conversion to tenofovir (TFV), is mainly eliminated by glomerular filtration and active tubular secretion. The major adverse effect of tenofovir is nephrotoxicity; however, the exact mechanism remains poorly understood. In this study, the ATP-binding cassette subfamily C member 11 (ABCC11; multidrug resistance protein 8 [MRP8]) transporter, which is abundant in proximal tubular cells, was demonstrated to act as an efflux transporter of tenofovir. Real-time PCR (RT-PCR) and indirect immunofluorescence assays were used to determine MRP8 overexpression in a continuous cell line. Tenofovir accumulations were assessed by cytotoxicity, cellular transport, and vesicular uptake assays. Substrate specificity was confirmed using MK-571, an MRP-specific inhibitor, and methotrexate, which served as a known substrate. Intracellular and intravesicular concentrations of tenofovir were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The 50% cytotoxic concentration (CC50) of TDF in MRP8-overexpressing cells was 4.78 times higher than that of parental cells. Transport assays also showed that the intracellular accumulation of tenofovir in MRP8-overexpressing cells was 55 times lower than that in parental cells and was partly reversed by MK-571. Similarly, an "inside-out" vesicular uptake assay, using Sf9 inverted membrane vesicles to allow measuring of accumulation of the substrates into the vesicles, demonstrated a higher intravesicular concentration of tenofovir in MRP8-overexpressing vesicles than in Sf9 insect control vesicles. These effects were effectively reversed by increasing concentrations of the specific inhibitor MK-571. In conclusion, tenofovir is a new substrate of the MRP8 transporter. An alteration in the activity of this efflux pump may increase the intracellular accumulation of tenofovir in proximal renal tubular cells.

Vero-Toxigenic Escherichia Coli (VTEC): To Sum up all we know

Verotoxigenic Escherichia coli renamed after detection of production ability of cytotoxin which kills Vero cells, a continuous cell line derived from the kidney of the African green monkey (Chlorocebus sp). These toxins are proved to cause severe diseases in humans. As an emerging food borne pathogen group, VTEC infection may lead to life threatening haemolytic-uremic syndrome (HUS).

An explant of heifer mammary gland to study the immune response of the organ

Continuous or primary epithelial cell lines have been extensively used to study the mammary gland immune response, but they are constituted by a single cell population. Our aim was to test whether an explant of heifer gland, where the tissue structure is maintained, might be a valid model to investigate the innate immune response to infection. The study was carried out on 2mm3-sections of heifer udders, in 2 consecutive trials, using LPS or LTA in the first trial and two different concentrations of Staphylococcus aureus (Staph. aureus) in the second. Treated and untreated sections were collected after 1h, 3h and 6h incubation; in the first trial, a final time-point at 18h was considered. The mRNA expression of TNFα, IL-1β, IL-6, IL-8 and LAP was analyzed by quantitative real-time PCR. Histological examination showed well-preserved morphology of the tissue, and apoptosis only showed a slight, not significant increase throughout the experiment. IL-1β and IL-6 were significantly up-regulated, in response to LPS or Staph. aureus, while TNF-α and IL-8 significantly increased only under LPS treatment. LAP expression showed a significant late increase when stimulated by LPS. The immunochemical staining of the sections demonstrated a higher number of T lymphocytes within the alveolar epithelium, in comparison with interstitial localization. Since the explants belonged to pubertal non-pregnant heifers, T cells may be regarded as resident cells, suggesting their participation in the regulation of mammary homeostasis. Therefore, applying our model would give new insights in the investigation of udder pathophysiology.

Cell lines authentication and mycoplasma detection as minimun quality control of cell lines in biobanking

Establishment of continuous cell lines from human normal and tumor tissues is an extended and useful methodology for molecular characterization of cancer pathophysiology and drug development in research laboratories. The exchange of these cell lines between different labs is a common practice that can compromise assays reliability due to contamination with microorganism such as mycoplasma or cells from different flasks that compromise experiment reproducibility and reliability. Great proportions of cell lines are contaminated with mycoplasma and/or are replaced by cells derived for a different origin during processing or distribution process. The scientific community has underestimated this problem and thousand of research experiment has been done with cell lines that are incorrectly identified and wrong scientific conclusions have been published. Regular contamination and authentication tests are necessary in order to avoid negative consequences of widespread misidentified and contaminated cell lines. Cell banks generate, store and distribute cell lines for research, being mandatory a consistent and continuous quality program. Methods implementation for guaranteeing both, the absence of mycoplasma and authentication in the supplied cell lines, has been performed in the Andalusian Health System Biobank. Specifically, precise results were obtained using real time PCR detection for mycoplasma and 10 STRs identification by capillary electrophoresis for cell line authentication. Advantages and disadvantages of these protocols are discussed.

In vitro differentiation of neural stem cells derived from human olfactory bulb into dopaminergic-like neurons.

This study describes a new accessible source of neuronal stem cells that can be used in Parkinson's disease cell transplant. The human olfactory bulb contains neural stem cells (NSCs) that are responsible for neurogenesis in the brain and the replacement of damaged cellular components throughout life. NSCs are capable of differentiating into neuronal and glial cells. We isolated NSCs from the olfactory bulb of brain-death donors and differentiated them into dopaminergic neurons. The olfactory bulb tissues obtained were cultured in Dulbecco's modified Eagle's medium/nutrient mixture F12, B27 supplemented with basic fibroblast growth factor, epidermal growth factor and leukemia inhibitory factor. The NSCs and proliferation markers were assessed. The multipotentiality of olfactory bulb NSCs was demonstrated by their capacity to differentiate into neurons, oligodendrocytes and astrocytes. To generate dopaminergic neurons, olfactory bulb NSCs were differentiated in neurobasal medium, supplemented with B27, and treated with sonic hedgehog, fibroblast growth factor 8 and glial cell-derived neurotrophic factor from the 7th to the 21st day, followed by detection of dopaminergic neuronal markers including tyrosine hydroxylase and aromatic l-amino acid decarboxylase. The cells were expanded, established in continuous cell lines and differentiated into the two classical neuronal phenotypes. The percentage of co-positive cells (microtubule-associated protein 2 and tyrosine hydroxylase; aromatic l-amino acid decarboxylase and tyrosine hydroxylase) in the treated cells was significantly higher than in the untreated cells. These results illustrate the existence of multipotent NSCs in the adult human olfactory bulb that are capable of differentiating toward putative dopaminergic neurons in the presence of trophic factors. Taken together, our data encourage further investigations of the possible use of olfactory bulb NSCs as a promising cell-based therapeutic strategy for Parkinson's disease.

Initiation of primary cell cultures from embryonic Haemaphysalis bispinosa ticks

Tick cell cultures have been widely used as an important tool for the study of tick-associated microorganisms, specifically for medically important bacteria or viruses that may be difficult to isolate or culture in axenic conditions. In this study, primary embryonic tick cell cultures were initiated separately from each of the egg batches laid by 10 female ticks belonging to the hard tick genus Haemaphysalis. All cultures were maintained at 28°C. After 10 months, 4 healthy cultures were identified with the potential for developing into continuous tick cell lines. These cultures comprise large cells predominantly forming floating cell clumps with multicellular vesicles, which are morphologically similar to cell lines derived from the soft tick Ornithodoros moubata. Subculture has not yet been performed due to the low cell density at the time of writing. Amplification and sequencing of a fragment of the 16S rRNA gene from DNA extracted from the parent ticks showed 99%-100% similarity to published sequences of Haemaphysalis bispinosa. This is the first report of the initiation of embryonic cell cultures from Haemaphysalis ticks found in Malaysia. Such tick cell cultures will be useful for studies of tick-borne pathogens in this region, where recent studies have shown that Haemaphysalis ticks are highly represented and harbor medically important bacteria.

Efficacy evaluation of the C-strain-based vaccines against the subgenotype 2.1d classical swine fever virus emerging in China

Classical swine fever (CSF) is a devastating infectious disease of pigs caused by classical swine fever virus (CSFV). The disease has been controlled following extensive vaccination with the lapinized attenuated vaccine C-strain for decades in China. However, frequent CSF outbreaks occurred recently in a large number of C-strain-vaccinated pig farms in China and a new subgenotype 2.1d of CSFV has been reported to be responsible for the outbreaks. Here we analyzed the molecular variations and antigenic differences among the C-strain-based commercial vaccines of different origins from different manufacturers in China, and reevaluated the vaccines against the emerging subgenotype 2.1d strain of CSFV. The results showed that the C-strain adapted to the continuous ST cell line (CST) contain a unique M290K variation on the E2 protein, compared to those of primary BT cells (CBT) or rabbit origin (CRT) and the traditional C-strain sequences available in the GenBank database. Serum neutralization test revealed the antigenic differences between CST and CBT or CRT. Notably, the neutralizing titers of porcine anti-C-strain sera against the CSFV isolate of subgenotype 2.1d were significantly lower than those against C-strain or Shimen strain. The C-strain-vaccinated, subgenotype 2.1d HLJZZ2014 strain-challenged pigs did not show any clinical signs and all survived. However, these pigs displayed mild pathological and histological lesions, and the CSFV viral RNA was detected in the various tissue and blood samples. Taken together, the C-strain-based vaccines of different origins showed molecular variations and antigenic differences, and could provide clinical but not pathological and virological protection against the subgenotype 2.1d CSFV emerging in China. Further investigation is needed to comprehensively assess the efficacy of C-strain of different doses against the subgenotype 2.1d CSFV.

Establishment and Characterization of an SV40 Large T Antigen-Transduced Porcine Colonic Epithelial Cell Line

Continuous cell lines have become indispensable tools that have enabled investigations into cellular mechanisms by increasing experimental reproducibility and sample availability, and decreasing the use of experimental animals. To facilitate studies of epithelial barrier function of the porcine colon, we aimed to establish an epithelial cell line with an extended replicative capacity. Cells were isolated from the proximal colon of a 3-week-old piglet and transduced using a recombinant retroviral vector construct containing the simian virus 40 large T antigen (SV40 TAg). We established a clonal epithelial cell line, referred to as PoCo83-3, that stably expressed the SV40 TAg, verified at mRNA and protein levels. PoCo83-3 showed epithelial cell-specific features, such as cobblestone-like morphology, dome structure formation, the presence of apical microvilli, and the expression of keratin 18, E-cadherin and the tight junction-associated proteins zonula occludens-1, occludin, and claudin-1. To validate PoCo83-3 as an in vitro model in epithelial barrier research, proinflammatory cytokine-inducible alterations in barrier integrity were demonstrated by incubating the cells with TNF-α and IFN-γ for 48 h. These cytokine treatments promoted a decreased transepithelial electrical resistance. In summary, PoCo83-3 exhibited an extended life span and a differentiated phenotype while maintaining epithelial characteristics. Based on these results, we present this cell line as a valuable in vitro model for investigations of epithelial barrier function in the porcine colon.

Influenza vaccines manufacturing in continuous cell lines: problems and solutions

ABSTRACT In order to decrease the morbidity and mortality caused by seasonal influenza outbreaks, several hundred million vaccine doses are produced worldwide each year. The predominant substrate for the production of the influenza vaccine today is fertilized hen’s eggs. The substitution of the technology based on living organisms by the cell culture-based process offers many advantages, including easier scalability and reduced dependence on the availability of eggs. The African green monkey kidney and Madin Darby canine kidney cell lines support the efficient growth of influenza viruses of different subtypes and, therefore, are considered to be the two most promising alternative substrates for the production of the human influenza vaccine. However, the pH of endosomes in both of these cell lines is higher than the pH essential for triggering a conformational change of the hemagglutinin (HA) of human influenza viruses, which enables the viral-cellular membrane fusion. This mismatch gives rise to mutations in the HA that lead to an increase of the optimum pH of HA conformational change. As of a result of these mismatches, the HA, and consequently the whole virus, has reduced stability to low pH and elevated temperatures. The production of a vaccine from less stable virus will lead to an elevated HA content in the low pH conformation that can affect the safety, potency, infectivity, and protective efficacy of the final inactivated and live attenuated influenza vaccines. The main limitations of the cell line-based influenza vaccine technology and the possibilities to preserve the viral stability over the course of influenza vaccine production are discussed in the review.

Production of Camel Pox Vaccine in the Sudan

Camels are important animals adapted to hot and arid environments, they represent an income to owner in rural areas hence, helping to upgrade the livelihood and alleviate poverty among people in these areas. Sudan ranks number two in camel’s population in the world, with more than 4 million heads of camel. The camel pox disease causes high morbidity (100%) and high mortality (10%-50%), especially in young animals. Effective control programs such as sanitary measures, quarantine of infected areas, restriction of camel movement, provision of clean drinking water and avoidance of skin abrasions are effective control measurements, unfortunately these methods are not applicable in Sudan owing to the migratory pattern of camel breeding in the country and difficulty to reach camels in remote area especially during the rainy season. In addition there is no treatment for camel pox disease, therefore, vaccination is regarded as the most effective tool for disease prevention. Few camel poxes both inactivated and live-attenuated vaccines are available in a few countries. Live vaccines are the best method as they provide long-lasting immunity. In the present study camel pox vaccine seed virus was obtained from African Union- Pan African Veterinary Vaccine Centre (AU.PANVAC) and successfully reproduced and evaluated in local lab condition and experimentally investigated in local host animals and environment. The vaccine was produced according to OIE manual and subjected for identity, safety, potency, efficacy and immunogenicity tests. The identity test for the vaccine master seeds was fulfilled as the first step using Varity of identification methods, Agar Gel Precipitation Test (AGPT), Virus Neutralization Test (VNT) and Polymarise Chain Reaction (PCR) tests. Moreover, sterility test was carried out for bacterial and fungal contaminations. The vaccine was produced using seed lot system i.e. Master Seed (MS) was used to produce the Working Seed Bank (WSB) from which an experimental batch was produced. All the production processes were carried out in an aseptic conditions using continuous cell line of African Green Monkey kidney cells (VERO). The safety profile for working seed lot was evaluated in host animal by inoculation of 10 camels obtained from local market with different age groups, animals were inoculated subcutaneously with 10 times the recommended field dose i.e.104 Tissue culture Infected Dose of fifty (TCID50) (OIE,2014). The immunogenicity of candidate vaccine was assessed in 20 healthy camels consequently, 14 camels were vaccinated by 103 TCID50 by S/C(OIE,2014) route and 6 were inoculated by phosphate buffer saline (PBS) and kept as un vaccinated control. The vaccine was safe and the inoculated animals remained healthy without any adverse reactions, neither signs of illness nor a rise in rectal temperature were recorded for up to 6 weeks post vaccination. The Abs mediated immune response i.e. immunogenicity was measured by serum neutralization test, the vaccinated camels demonstrated protective level of 5 (log2) Abs after 4 weeks post vaccination. While, no production of antibodies detected in the sera of the control group. After 4 weeks post vaccination all the vaccinated and control groups were challenged subcutaneously by local isolate of camel pox using 105.6 TClD50 / animal. Only the unvaccinated control group developed very severe clinical signs and fever reached 40oc with generalized and localized camel pox lesions, while the vaccinated groups withstood the test without death or clinical signs.