Madin-Darby Canine Kidney Cell Line Research Articles

Evaluation of blood–brain barrier and blood–cerebrospinal fluid barrier permeability of 2-phenoxy-indan-1-one derivatives using in vitro cell models

2-Phenoxy-indan-1-one derivatives (PIOs) are a series of novel central-acting cholinesterase inhibitors for the treatment of Alzheimer's disease (AD). The adequate distribution of PIOs to the central nervous system (CNS) is essential for its effectiveness. However, articles related with their permeability in terms of CNS penetration across the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB) have not been found. This study was undertaken to evaluate the in vitro BBB and BCSFB transport of PIOs using Madin-Darby canine kidney (MDCK), MDCK-MDR1 and Z310 cell line models. As a result, the transepithelial transport of PIOs did not differ between MDCK and MDCK-MDR1, and the result suggested that PIOs were not substrates for P-gp, which means that multidrug resistance (MDR) function would not affect PIOs absorption and brain distribution. High permeability of PIOs in Z310 was found and it suggested that PIOs had high brain uptake potential. The experiment also showed that PIOs had inhibitory effects on the MDR1-mediated transport of Rhodamine123 with an IC50 value of 40-54 μM. And we suggested that 5,6-dimethoxy-1-indanone might be the pharmacophoric moiety of PIOs that interacts with the binding site of P-gp.

Activation of liver X receptors reduces CFTR-mediated Cl−transport in kidney collecting duct cells

Liver X receptors (LXRs) are transcription factors belonging to the nuclear receptor super family, which act as regulators of lipid and glucose metabolism. However, LXRs have been shown to regulate the function of transporters in the kidney, including the Na-Pi cotransporter, organic anion transporter, and epithelial Na(+) channel. In this report, we demonstrated the ability of LXR ligands, both endogenous [22 (R)-hydroxycholesterol] and synthetic (T0901317 and GW3965), to reduce CFTR-mediated Cl(-) secretion in a type I Madin-Darby canine kidney (MDCK) cell line and in murine primary inner medullary collecting duct (IMCD) cells, based on measurements of [Arg(8)]-vasopressin-induced Cl(-) current. However, treatment of MDCK cell monolayers with 5 μM T0901317 for 24 h did not alter ouabain-senstive current or Na(+)-K(+)-ATPase-α protein content. Furthermore, basolateral membranes permeabilization of MDCK cell monolayers still resulted in a decrease in apical Cl(-) current stimulated by both [Arg(8)]-vasopressin and 8-cholorophenyl-thio-cAMP, indicating that the factor(s) encoded by the target gene(s) of agonist-activated LXRs might be located at the apical membrane. Western blot analysis revealed that inhibition of Cl(-) secretion occurred via a decrease in CFTR protein, which was not due to downregulation of its mRNA expression. In addition, both synthetic LXR agonists significantly retarded the growth of forskolin-induced cysts formed in MDCK cells cultured in collagen gel. This is the first evidence showing that ligand-activated LXRs are capable of downregulating CFTR-mediated Cl(-) secretion of kidney cells and of retarding cyst growth in a MDCK cell model.

Long term culture of MDCK strains alters chromosome content

BackgroundThe Madin-Darby Canine Kidney (MDCK) cell line and its different strains are widely used as models for studying epithelial simple polarity. Recently Dukes et al. [BMC Cell Biology 12:43, 2011] provided a useful guide to the different MDCK strains, with a directory of where to buy them. The present work focused on chromosome content of MDCK cells, a parameter often disregarded by researchers working with these cells.FindingsUsing a general and reliable method for obtaining high yield of metaphasic preparations, the chromosome content of MDCK, MDCK I, MDCK II obtained from reliable sources was determined after maintaining them in culture for various periods of time. Within two months significant changes were observed in the range and the mean number of chromosomes of MDCK I and MDCK II cells. MDCK II cells routinely cultured in six different laboratories were also examined. In some of these cultures the cells have considerably drifted as shown by a high scattering in their number of chromosomes.ConclusionThese results entitle me to encourage researchers using MDCK cells obtained from reliable sources, to determine their chromosomal content upon receipt, to check this content after several passages, to use this feature to follow the possible drift of these cells, and finally to avoid working with cells maintained for more than two months in culture.

Immunolocalization and translocation of aquaporin-5 water channel in sweat glands

BackgroundAquaporin-5 (AQP5) is a member of the water channel protein family. Although AQP5 was shown to be present in sweat glands, the presence or absence of regulated intracellular translocation of AQP5 in sweat glands remained to be determined. ObjectiveWe investigated whether AQP5 in sweat glands translocated during sweating, and also sought to determine the intracellular signal that triggers this translocation. MethodsImmunofluorescent analyses of AQP5 in mouse and human sweat glands were performed. Madin-Darby Canine kidney (MDCK) cell lines stably expressing human AQP5 were generated, and the regulated translocation of AQP5 in the polarized cells was assessed by immunofluorescent analysis and biotinylation assays. ResultsAQP5 showed rapid translocation to the apical membranes during sweating. In human eccrine sweat glands, immunoreactive AQP5 was detected in the apical membranes and the intercellular canaliculi of secretory coils, and in the basolateral membranes of the clear cells. Treatment of human AQP5-expressing MDCK cells with calcium ionophore A23187 resulted in a twofold increase of AQP5 in the apical membranes within 5min. ConclusionThe regulated AQP5 translocation may contribute to sweat secretion by increasing the water permeability of apical plasma membranes of sweat glands.

Trafficking of the IKs‐Complex in MDCK Cells: Site of Subunit Assembly and Determinants of Polarized Localization

The voltage-gated potassium channel KV 7.1 is regulated by non-pore forming regulatory KCNE β-subunits. Together with KCNE1, it forms the slowly activating delayed rectifier potassium current IKs . However, where the subunits assemble and which of the subunits determines localization of the IKs -complex has not been unequivocally resolved yet. We employed trafficking-deficient KV 7.1 and KCNE1 mutants to investigate IKs trafficking using the polarized Madin-Darby Canine Kidney cell line. We find that the assembly happens early in the secretory pathway but provide three lines of evidence that it takes place in a post-endoplasmic reticulum compartment. We demonstrate that KV 7.1 targets the IKs -complex to the basolateral membrane, but that KCNE1 can redirect the complex to the apical membrane upon mutation of critical KV 7.1 basolateral targeting signals. Our data provide a possible explanation to the fact that KV 7.1 can be localized apically or basolaterally in different epithelial tissues and offer a solution to divergent literature results regarding the effect of KCNE subunits on the subcellular localization of KV 7.1/KCNE complexes.

H3N2 homeopathic influenza virus solution modifies cellular and biochemical aspects of MDCK and J774G8 cell lines

Influenza viruses cause highly contagious acute respiratory illnesses with significant mortality, especially among young children, elderly people, and individuals with serious medical conditions. This encourages the development of new treatments for human flu. Biotherapies are diluted solutions prepared from biological products compounded following homeopathic procedures. To develop a biotherapy prepared from the infectious influenza A virus (A/Aichi/2/68 H3N2) and to verify its invitro response. The ultradiluted influenza virus solution was prepared in the homeopathic dilution 30dH, it was termed Influenzinum RC. The cellular alterations induced by this preparation were analyzed by optical and electron microscopy, MTT and neutral red assays. Glycolytic metabolism (PFK-1) was studied by spectrophotometric assay. Additionally, the production of tumor necrosis factor-α (TNF-α) by J774.G8 macrophage cells was quantified by ELISA before and after infection with H3N2 influenza virus and treatment. Influenzinum RC did not cause cytotoxic effects but induced morphological alterations in Madin-Darby canine kidney (MDCK) cells. After 30 days, a significant increase (p<0.05) in mitosis rate was detected compared to control. MDCK mitochondrial activity was changed after treatment for 10 and 30 days. Treatment significantly diminished (p<0.05) PFK-1 activity. TNF-α in biotherapy-stimulated J774.G8 macrophages indicated a significant (p < 0.05) increase in this cytokine when the cell supernatant was analyzed. Influenzinum RC altered cellular and biochemical features of MDCK and J774G8 cells.

Environmental hyperosmolality regulates phospholipid biosynthesis in the renal epithelial cell line MDCK.

Hyperosmolality is a key signal for renal physiology. On the one hand, it contributes to the differentiation of renal medullary structures and to the development of the urinary concentrating mechanism. On the other, it is a stress factor. In both cases, hyperosmolality activates processes that require an adequate extension of cellular membranes. In the present work, we examined whether hyperosmolality regulates phospholipid biosynthesis, which is needed for the membrane biogenesis in the renal epithelial cell line Madin-Darby canine kidney (MDCK). Because phospholipids are the structural determinants of all cell membranes, we evaluated their content, synthesis, and regulation in MDCK cultures subjected to different hyperosmotic concentrations of NaCl, urea, or both. Hyperosmolality increased phospholipid content in a concentration-dependent manner. Such an effect was exclusively due to changes in NaCl concentration and occurred at the initial stage of hyperosmolar treatment concomitantly with the expression of the osmoprotective protein COX-2. The hypertonic upregulation of phosphatidylcholine (PC) synthesis, the main constituent of all cell membranes, involved the transcriptional activation of two main regulatory enzymes, choline kinase (CK) and cytidylyltransferase α (CCTα) and required ERK1/2 activation. Considering that physiologically, renal medullary cells are constantly exposed to high and variable NaCl, these findings could contribute to explaining how renal cells could maintain cellular integrity even in a nonfavorable environment.

Cell membrane morphology analysis using an infrared sensor system

In this paper, we report on the CH2-symmetric to CH2-antisymmetric stretch ratio as a method to determine membrane morphology alterations in mammalian cells. Cell membrane morphology is an important parameter for tumour development studies and for determining the lowest effective dose of antitumour agents, reducing the occurrence of side effects. For this study a normal epithelial kidney cell line and three human melanoma cell lines were investigated. The epithelial kidney cell line Madin-Darby Canine Kidney was exposed to the cholesterol reduction agent methyl-β-cyclodextrin. The cholesterol reduction changes the cell membrane morphology and the plasma membrane of the cells become less rigid. A thin layer chromatography analysis was performed into test the efficiency of the cholesterol reduction method.The antitumour agent cisplatin is known to cause the opposite effect and makes the cell membrane more rigid. In order to investigate the cell membrane morphology change three human melanoma cell lines were treated with cisplatin.For the CH2-stretch ratio measurements we used a self-designed infrared sensor system, which has the potential to be further developed in a point-of-care instrument. With the sensor system the morphology changes due to cholesterol reduction and cisplatin treatments were established.This study proves that the CH2-symmetric to CH2-antisymmetric stretch ratio can be successfully applied for label-free cell membrane morphology analysis.

The Madin-Darby canine kidney cell culture derived influenza A/H5N1 vaccine: A Phase I trial in Taiwan

Avian H5N1 influenza has caused human infections globally and has a high mortality rate. Rapid production of effective vaccines is needed. A phase 1, randomized, observer-blinded clinical trial was conducted to examine the safety and immunogenicity of an inactivated whole virion vaccine against the influenza A/H5N1 virus produced from the Madin-Darby canine kidney (MDCK) cell line. Participants were randomized to four groups and administered two intramuscular doses of vaccine containing 3 μg hemagglutinin (HA), 3 μg HA with 300 μg aluminum phosphate (AlPO4), 6 μg HA, and 6 μg HA with 300 μg AlPO4, respectively, at two visits, 21 days apart. Serum hemagglutination inhibition (HAI) and neutralizing antibody levels were determined at baseline and on Days 21 and 42. Sixty healthy individuals were enrolled. The neutralization assay showed a significant immune response in the 6 μg with ALPO4 group on Day 42 compared to pre-vaccination levels (11.32±9.77 vs. 4.00±0, p=0.02). The adjuvant effect in neutralization assay was also significant on Day 42 in the 6 μg group (4.52±1.94 without adjuvant vs. 11.32±9.77 with adjuvant, p=0.02). HAI assay also showed an aluminum adjuvant-induced increasing trend in HAI geometric mean titer on Day 42 in the 3 μg and 6 μg groups (6.02 versus 8.20, p=0.05 and 5.74 versus 8.21, p=0.14). The most frequent adverse event was local pain (20% to 60%). There were no vaccine-related severe adverse effects. MDCK cell line-derived H5N1 vaccine was well tolerated. It is necessary to investigate further the immunogenicity of higher antigen doses and the role of aluminum adjuvant in augmenting the effect of the vaccine.

Evaluation of tumorigenic potential of high yielding cloned MDCK cells for live-attenuated influenza vaccine using in vitro growth characteristics, metastatic gene expression and in vivo nude mice model

Several mammalian cell lines, including Madin–Darby canine kidney (MDCK) cells have been approved by regulators for manufacturing of human vaccines. A new MDCK 9B9-1E4 cloned cell line has been created which is capable of producing live attenuated influenza vaccine (LAIV) with high yield. This cell line was shown to be non tumorigenic in eight week old adult athymic nude mouse model. This property is desirable for vaccine production and is unique to this cell line and is not known to be shared by other MDCK cell lines that are currently used for vaccine production. This significant difference in tumorigenic phenotype required further characterization of this cell line to ensure its safety for use in vaccine production. This is particularly important for LAIV production where it is not possible to incorporate a virus inactivation and/or removal step during manufacturing. Characterization of this cell line included extensive adventitious agent testing, tumorigenicity and oncogenicity assessment studies. Here, we describe the development of tumorigenic MDCK cell lines for use as positive controls and in vitro methods to aid in the evaluation of the tumorigenicity of MDCK 9B9-1E4 cloned cells. Tumorigenic MDCK cells were successfully generated following Hras and cMyc oncogene transfection of MDCK 9B9-1E4 cloned cells. In this study we demonstrate the lack of tumorigenic potential of the MDCK 9B9-1E4 cloned cell line in adult athymic nude mice model.

Sotalol permeability in cultured-cell, rat intestine, and PAMPA system.

To clarify sotalol's classification in the BCS versus BDDCS systems through cellular, rat everted sac and PAMPA permeability studies. Studies were carried out in Madin Darby canine kidney (MDCK) and MDR1-transfected MDCK (MDCK-MDR1) cell lines, rat everted gut sacs and the Parallel Artificial Membrane Permeability Assay (PAMPA) system. Three-hour transport studies were conducted in MDCK cell lines (with apical pH changes) and MDCK-MDR1 cells (with and without the P-glycoprotein inhibitor GG918); male Sprague-Dawley rats (300~350g) were used to prepare everted sacs. In the PAMPA studies, drug solutions at different pH's were dosed in each well and incubated for 5h. Samples were measured by LC-MS/MS, or liquid scintillation counting and apparent permeability (P(app)) was calculated. Sotalol showed low permeability in all of the cultured-cell lines, everted sacs and PAMPA systems. It might be a border line P-glycoprotein substrate. The PAMPA study showed that sotalol's permeability increased with a higher apical pH, while much less change was found in MDCK cells. The low permeability rate for sotalol correlates with its Class 3 BDDCS assignment and lack of in vivo metabolism.

Biocompatibility Of Synthesised Nano-Porous Anodic Aluminium Oxide Membranes For Use As A Cell Culture Substrate For Madin-Darby Canine Kidneys Cells: A Preliminary Study

In this study we investigate for the first time the biomedical potential of using a membrane made from anodic aluminium oxide (AAO) for culturing the Madin-Darby Canine Kidney (MDCK) epithelial cell line. Nano-porous aluminium oxide membranes exhibit interesting properties such as high porosity, which allows the exchange of molecules and nutrients across the membrane and can be made with highly specific pore sizes that can be preselected by adjusting the controlling parameters of a temperature controlled two-step anodization process. The cellular response and interactions of the MDCK cell line with the synthesised nano-porous AAO membrane, a commercially available membrane and a glass control were assessed by investigating cell adhesion, morphology and proliferation.

Zonula occludens-1 and -2 regulate apical cell structure and the zonula adherens cytoskeleton in polarized epithelia

The structure and function of both adherens (AJ) and tight (TJ) junctions are dependent on the cortical actin cytoskeleton. The zonula occludens (ZO)-1 and -2 proteins have context-dependent interactions with both junction types and bind directly to F-actin and other cytoskeletal proteins, suggesting ZO-1 and -2 might regulate cytoskeletal activity at cell junctions. To address this hypothesis, we generated stable Madin-Darby canine kidney cell lines depleted of both ZO-1 and -2. Both paracellular permeability and the localization of TJ proteins are disrupted in ZO-1/-2-depleted cells. In addition, immunocytochemistry and electron microscopy revealed a significant expansion of the perijunctional actomyosin ring associated with the AJ. These structural changes are accompanied by a recruitment of 1-phosphomyosin light chain and Rho kinase 1, contraction of the actomyosin ring, and expansion of the apical domain. Despite these changes in the apical cytoskeleton, there are no detectable changes in cell polarity, localization of AJ proteins, or the organization of the basal and lateral actin cytoskeleton. We conclude that ZO proteins are required not only for TJ assembly but also for regulating the organization and functional activity of the apical cytoskeleton, particularly the perijunctional actomyosin ring, and we speculate that these activities are relevant both to cellular organization and epithelial morphogenesis.

Development of a new permeability assay using low‐efflux MDCKII cells

Permeability is an important property of drug candidates. The Madin-Darby canine kidney cell line (MDCK) permeability assay is widely used and the primary concern of using MDCK cells is the presence of endogenous transporters of nonhuman origin. The canine P-glycoprotein (Pgp) can interfere with permeability and transporter studies, leading to less reliable data. A new cell line, MDCKII-LE (low efflux), has been developed by selecting a subpopulation of low-efflux cells from MDCKII-WT using an iterative fluorescence-activated cell sorting technique with calcein-AM as a Pgp and efflux substrate. MDCKII-LE cells are a subpopulation of MDCKII cells with over 200-fold lower canine Pgp mRNA level and fivefold lower protein level than MDCKII-WT. MDCKII-LE cells showed less functional efflux activity than MDCKII-WT based on efflux ratios. Notably, MDCKII-MDR1 showed about 1.5-fold decreased expression of endogenous canine Pgp, suggesting that using the net flux ratio might not completely cancel out the background endogenous transporter activities. MDCKII-LE cells offer clear advantages over the MDCKII-WT by providing less efflux transporter background signals and minimizing interference from canine Pgp. The MDCKII-LE apparent permeability values well differentiates compounds from high to medium/low human intestinal absorption and can be used for Biopharmaceutical Classification System. The MDCKII-LE permeability assay (4-in-1 cassette dosing) is high throughput with good precision, reproducibility, robustness, and cost-effective.

AMP‐Activated Protein Kinase Downregulates Kv7.1 Cell Surface Expression

The potassium channel Kv7.1 is expressed in the heart, where it contributes to the repolarization of the cardiac action potential. Additionally, Kv7.1 is expressed in epithelial tissues playing a role in salt and water transport. We recently demonstrated that surface-expressed Kv7.1 is internalized in response to polarization of the epithelial Madin-Darby canine kidney (MDCK) cell line and that this was mediated by activation of protein kinase C (PKC). In this study, the pathway downstream of PKC, which leads to internalization of Kv7.1 upon cell polarization, is elucidated. We show by confocal microscopy that Kv7.1 is endocytosed upon initiation of the polarization process and sent for degradation by the lysosomal pathway. The internalization could be mimicked by pharmacological activation of the AMP-activated protein kinase (AMPK) using three different AMPK activators. We demonstrate that the downstream effector of AMPK is the E3 ubiquitin ligase Nedd4-2. Additionally, we show that AMPK activation results in a downregulation of Kv7.1 currents in Xenopus oocytes through a Nedd4-2-dependent mechanism. In summary, surface-expressed Kv7.1 channels are endocytosed and sent for degradation in lysosomes by an AMPK-mediated activation of Nedd4-2 during the initial phase of the MDCK cell polarization process.

New Strategies for the Development of H5N1 Subtype Influenza Vaccines

The emergence and spread of highly pathogenic avian influenza (H5N1) viruses among poultry in Asia, the Middle East, and Africa have fueled concerns of a possible human pandemic, and spurred efforts towards developing vaccines against H5N1 influenza viruses, as well as improving vaccine production methods. In recent years, promising experimental reverse genetics-derived H5N1 live attenuated vaccines have been generated and characterized, including vaccines that are attenuated through temperature-sensitive mutation, modulation of the interferon antagonist protein, or disruption of the M2 protein. Live attenuated influenza virus vaccines based on each of these modalities have conferred protection against homologous and heterologous challenge in animal models of influenza virus infection. Alternative vaccine strategies that do not require the use of live virus, such as virus-like particle (VLP) and DNA-based vaccines, have also been vigorously pursued in recent years. Studies have demonstrated that influenza VLP vaccination can confer homologous and heterologous protection from lethal challenge in a mouse model of infection. There have also been improvements in the formulation and production of vaccines following concerns over the threat of H5N1 influenza viruses. The use of novel substrates for the growth of vaccine virus stocks has been intensively researched in recent years, and several candidate cell culture-based systems for vaccine amplification have emerged, including production systems based on Madin-Darby canine kidney, Vero, and PerC6 cell lines. Such systems promise increased scalability of product, and reduced reliance on embryonated chicken eggs as a growth substrate. Studies into the use of adjuvants have shown that oil-in-water-based adjuvants can improve the immunogenicity of inactivated influenza vaccines and conserve antigen in such formulations. Finally, efforts to develop more broadly cross-protective immunization strategies through the inclusion of conserved influenza virus antigens in vaccines have led to experimental vaccines based on the influenza hemagglutinin (HA) stem domain. Such vaccines have been shown to confer protection from lethal challenge in mouse models of influenza virus infection. Through further development, vaccines based on the HA stem have the potential to protect vaccinated individuals against unanticipated pandemic and epidemic influenza virus strains. Overall, recent advances in experimental vaccines and in vaccine production processes provide the potential to lower mortality and morbidity resulting from influenza infection.

New Evidence of a Dihydropyridine-Activated Cationic Channel in the MDCK Cell Line

Newborn rat distal cells express an apical Ca²⁺ channel activated by dihydropyridine drugs. Similarly, in Madin-Darby canine kidney (MDCK) cells, nifedipine increased Ca²⁺_i in a concentration-dependent manner (IC₅₀ = 4 µM) in fura-2-loaded cells. Response to nifedipine was abolished by EGTA, suggesting that it depends on extracellular calcium. Ca²⁺ channel antagonist isradipine and agonist BayK8644 increased Ca²⁺_i indicating that this effect is related to the dihydropyridine group. Diltiazem (20 µM) and gadolinium (200 µM) decreased the nifedipine effect (62 and 43%, respectively). Lanthanum (100 µM) did not change the response. Valinomycin clamping of the membrane potential did not modify nifedipine-induced increment, indicating that it was unrelated to potassium fluxes. We performed whole cell clamp experiments in MDCK cells maintained at –50 mV with perfusion solution containing 10 mM CaCl₂. Nifedipine (20 µM) induced an increase in current (1.2 ± 0.3 nA), which was partially inhibited by Gd³⁺. No significant current was induced by nifedipine in the presence of 0.5 mM EGTA. To determine the effects of nifedipine on the membrane potential, we performed oxonol fluorescence experiments. The addition of nifedipine or Bay K8644 induced depolarization, highly dependent on external sodium. Nifedipine (20 µM) induced depolarization of 6.9 ± 0.8 mV (n = 21). EC₅₀ to nifedipine was in the 10 µM range. We conclude that MDCK cells exhibit a dihydropyridine-activated cationic channel.

Kv7.1 surface expression is regulated by epithelial cell polarization

The potassium channel K(V)7.1 is expressed in the heart where it contributes to the repolarization of the cardiac action potential. In addition, K(V)7.1 is expressed in epithelial tissues where it plays a role in salt and water transport. Mutations in the kcnq1 gene can lead to long QT syndrome and deafness, and several mutations have been described as trafficking mutations. To learn more about the basic mechanisms that regulate K(V)7.1 surface expression, we have investigated the trafficking of K(V)7.1 during the polarization process of the epithelial cell line Madin-Darby Canine Kidney (MDCK) using a modified version of the classical calcium switch. We discovered that K(V)7.1 exhibits a very dynamic localization pattern during the calcium switch. When MDCK cells are kept in low calcium medium, K(V)7.1 is mainly observed at the plasma membrane. During the first hours of the switch, K(V)7.1 is removed from the plasma membrane and an intracellular accumulation in the endoplasmic reticulum (ER) is observed. The channel is retained in the ER until the establishment of the lateral membranes at which point K(V)7.1 is released from the ER and moves to the plasma membrane. Our data furthermore suggest that while the removal of K(V)7.1 from the cell surface and its accumulation in the ER could involve activation of protein kinase C, the subsequent release of K(V)7.1 from the ER depends on phosphoinositide 3-kinase (PI3K) activation. In conclusion, our results demonstrate that K(V)7.1 surface expression is regulated by signaling mechanisms involved in epithelial cell polarization in particular signaling cascades involving protein kinase C and PI3K.

Ultrastructural alterations induced by Δ24(25)-sterol methyltransferase inhibitors on Trichomonas vaginalis

Trichomonas vaginalis is an important human parasite that causes trichomoniasis, a cosmopolitan sexually transmitted disease. Currently, the treatment of choice for T. vaginalis infections is metronidazole. The increase in metronidazole-resistant parasites and undesirable side effects of this drug make the search for alternative chemotherapeutic approaches a priority for the management of trichomoniasis. Here, the antiproliferative and ultrastructural effects of sterol biosynthesis inhibitors against T. vaginalis were investigated. It was found that 22,26-azasterol (5 μM) and 24(R,S),25-epiminolanosterol (10 μM), known inhibitors of Δ(24(25))-sterol methyltransferase, exhibited antiproliferative effects on T. vaginalis trophozoites cultured in vitro. Morphological analyses showed that azasterols induced changes in the ultrastructure of T. vaginalis. The most significant alterations were (1) membrane blebbing and disruption, (2) wrinkled cells and (3) the formation of cell clusters. In addition, autophagic vacuoles, Golgi duplication arrest, an abnormal Golgi enlargement and damaged hydrogenosomes were also observed. Nonspecific cytotoxicity assays using the cultured mammalian cell lines Madin-Darby canine kidney cells showed no effect of the azasterols on the viability and proliferation of these cells at a concentration that significantly inhibited the proliferation of T. vaginalis, indicating a selective antiparasitic action. Taken together, these results suggest that azasterols could be important compounds in the development of novel chemotherapeutic approaches against T. vaginalis.

Adaptation of a Madin–Darby canine kidney cell line to suspension growth in serum-free media and comparison of its ability to produce avian influenza virus to Vero and BHK21 cell lines

Madin–Darby canine kidney (MDCK) cells are currently considered for influenza vaccine manufacturing. A drawback of these cells is their anchorage dependent growth, which greatly complicates process scale-up. In this paper a novel MDCK cell line (MDCK-SFS) is described that grows efficiently in suspension and retained high expression levels of both α-2,6 and α-2,3 sialic acid receptors, which bind preferably to human and avian influenza viruses, respectively. The production of avian influenza virus by BHK21, Vero and MDCK-SFS cell lines was compared. Although BHK21 cells consisted of two populations, one of which lacks the α-2,3 receptor, they supported the replication of two influenza strains to high titres. However, BHK21 cells are generally not applicable for influenza production since they supported the replication of six further strains poorly. MDCK-SFS cells yielded the highest infectious virus titres and virus genome equivalent concentration for five of the eight influenza strains analyzed and the highest hemagglutination activity for all eight virus strains. Taken together with their suitability for suspension growth this makes the MDCK-SFS cell line potentially useful for large scale influenza virus production.