High Serum Medium Research Articles

BSBM-17 NOVEL VALIDATED PATIENT-DERIVED MODELS OF A RECTAL CANCER BRAIN METASTASIS

Abstract Colorectal adenocarcinomas (CRC) rarely metastasize to the brain, however, survival rates in patients with colorectal brain metastases (CBM) are short, with a median survival of only 4 months from diagnosis. Currently, no evidence-based treatment options against these rare brain metastases are available (PMC). Therefore, characterization of these tumors and development of targeted therapies are imperative. Optimal models for neoplasms heighten the notoriously low success rate of bench-to-bedside translation. Historically, brain cancer metastases models are established cell lines grown on plastic and transplanted as xenografts (Valiente; PMC7572582). Genomic drift, lack of genetic accuracy and heterogeneity, caused by serial passaging on plastic and in high-serum media, are frequent limitations of these models. Growth in defined media without serum and as 3-dimensional (3-D) spheroids or organoids is thought to improve the fidelity of cancer cell lines (Lee; PMID:16697959; Lo; PMC820864). Despite significant efforts of large collaborative projects (https://hcmi-searchable-catalog.nci.nih.gov/), 3-D rectal brain cancer metastasis models have not been reported. To address this unmet medical need, we developed 3-D (suspension, organoid) cultures of a rectal adenocarcinoma brain metastasis with moderate to focally poor differentiation. Next-generation sequencing, expression profiling, and immunohistochemistry were performed to validate our models. Our data confirmed that the PDO model contains tumor cells; pathogenic alterations and expression profiles in the models largely match that of the original tumor. Overall, we detected a significant phenotypic overlap with the original tumor sample. Spatial profiling and PDO-derived xenografts are in progress and will facilitate experimental therapeutics. Taken together, our models retained tumor heterogeneity and fidelity from the primary tumor, making this one of the first studies to establish 3-D patient-derived models for disease progression in rectal cancer brain metastases. Collectively, these models will be valuable translational tools for future exploration of targeted therapy, which remains absent in this disease process.

Bi-Functional Gold Nanorod-Protein Conjugates with Biomimetic BSA@Folic Acid Corona for Improved Tumor Targeting and Intracellular Delivery of Therapeutic Proteins in Colon Cancer 3D Spheroids.

Gold nanorods (AuNRs) remain well-developed inorganic nanocarriers of small molecules for a plethora of biomedical and therapeutic applications. However, the delivery of therapeutic proteins using AuNRs with high protein loading capacity (LC), serum stability, excellent target specificity, and minimal off-target protein release is not known. Herein, we report two bi-functional AuNR-protein nanoconjugates, AuNR@EGFP-BSAFA and AuNR@RNaseA-BSAFA, supramolecularly coated with folic acid-modified BSA (BSAFA) acting as biomimetic protein corona to demonstrate targeted cytosolic delivery of enhanced green fluorescent protein (EGFP) and therapeutic ribonuclease A enzyme (RNase A) in their functional forms. AuNR@EGFP-BSAFA and AuNR@RNaseA-BSAFA exhibit high LCs of ∼42 and ∼54%, respectively, increased colloidal stability, and rapid protein release in the presence of biological thiols. As a nanocarrier, AuNR@EGFP-BSAFA and AuNR@RNaseA-BSAFA show resistance to corona formation in high-serum media even after 24 h, guaranteeing a greater circulation lifetime. Folate receptor-targeting BSAFA on the AuNR surface facilitates the receptor-mediated internalization, followed by the release of EGFP and RNase A in HT29 cells. The green fluorescence dispersed throughout the cell's cytoplasm indicates successful cytosolic delivery of EGFP by AuNR@EGFP-BSAFA. AuNR@RNaseA-BSAFA-mediated therapeutic RNase A delivery in multicellular 3D spheroids of HT29 cells exhibits a radical reduction in the cellular RNA fluorescence intensity to 38%, signifying RNA degradation and subsequent cell death. The versatile nanoformulation strategy in terms of the anisotropic particle morphology, protein type, and ability for targeted delivery in the functional form makes the present AuNR-protein nanoconjugates a promising platform for potential application in cancer management.

Smad3 influences Smad2 expression via the transcription factor C/EBPα and C/EBPβ during bovine myoblast differentiation

Transforming growth factor β (TGFβ) has participated in a variety of cellular biological processes. Smad2 and Smad3 are equally important TGFβ downstream effectors in mediating TGFβ signals. However, genes involved in controlling the balance between these two signaling pathways are unknown. In this study, we showed that although Smad2 and Smad3 are structurally similar, with 89% amino acid sequence similarity in bovine, Smad3 significantly decreased Smad2 mRNA and protein expression during bovine myoblast differentiation, but not by binding on its promoter. Luciferase assays and electrophoretic mobility shift assays (EMSA) demonstrated that the transcription factors C/EBPα and C/EBPβ activate Smad2 promoter activity and expression under high serum medium (GM), whereas the opposite was observed under low serum medium (DM). Moreover, over-expression and interference assays revealed that Smad3 has a different effect on C/EBPα and C/EBPβ expression under GM versus DM conditions. After mutation of the C/EBPα and C/EBPβ binding sites, Smad3 had a reduced effect on Smad2 promoter activity. Therefore, these results demonstrated that Smad3 inhibits Smad2 expression via its transcription factors C/EBPα and C/EBPβ during bovine myoblast differentiation. This novel mechanism of the Smad2/3 genes may offer clues for further investigation of TGFβ signal function.

Efficient and high yield isolation of myoblasts from skeletal muscle

Skeletal muscle (SkM) regeneration relies on the activity of myogenic progenitors that reside beneath the basal lamina of myofibers. Here, we describe a protocol for the isolation of the SkM progenitors from young and old mice by exploiting their outgrowth potential from SkM explants on matrigel coated dishes in the presence of high serum, chicken embryo extract and basic fibroblast growth factor. Compared to other protocols, this method yields a higher number of myoblasts (10–20 million) by enabling the outgrowth of these cells from tissue fragments. The majority of outgrowth cells (~90%) were positive for myogenic markers such as α7-integrin, MyoD, and Desmin. The myogenic cell population could be purified to 98% with one round of pre-plating on collagen coated dishes, where differential attachment of fibroblasts and other non-myogenic progenitors separates them from myoblasts. Moreover, the combination of high serum medium and matrigel coating provided a proliferation advantage to myogenic cells, which expanded rapidly (~24 h population doubling), while non-myogenic cells diminished over time, thereby eliminating the need for further purification steps such as FACS sorting. Finally, myogenic progenitors gave rise to multinucleated myotubes that exhibited sarcomeres and spontaneous beating in the culture dish.

Knockdown of HIF-1α inhibits the proliferation and migration of outer root sheath cells exposed to hypoxia in vitro: An involvement of Shh pathway

AimsOuter root sheath (ORS) is a highly proliferative component of a hair follicle. This study is performed to investigate whether hypoxia-induced elevation of hypoxia-inducible factor (HIF)-1α, a transcriptional activator, contributes to the outgrowth of ORS cells in vitro. Main methodsHair follicles with intact ORS collected from 4-month old male American minks were cultured in normoxic or hypoxic condition (3% oxygen) for 7days. Primary ORS cells isolated from the mink hair follicles were exposed to hypoxia for 12, 24 or 48h, and their proliferation was analyzed with immunofluorescence assay using anti-proliferating cell nuclear antigen (PCNA) antibody. The migratory ability of ORS cells was detected via the transwell chamber. The endogenous HIF-1α was knocked down with its specific siRNA in ORS cells. Key findingsHypoxic exposure induced an elevation of HIF-1α in ex vivo cultured hair follicles. The mRNA and protein levels of sonic hedgehog (Shh), Shh receptor Patched 1, Smoothened and glioma-associated oncogene homologue 1 were upregulated. In vitro, hypoxia induced an increase in HIF-1α in ORS cells. Further, under hypoxic condition, the number of PCNA-positive cells was increased, and more cells migrated towards high serum media. Hypoxia-enhanced proliferation and migration of ORS cells were suppressed either by HIF-1α siRNA or by pharmacological inhibitors of Shh pathway, cyclopamine and GANT61. The activation of Shh pathway was attenuated in HIF-1α-silenced ORS cells under hypoxic condition. SignificanceOur work demonstrates a direct role of activated HIF-1/Shh biological axis in sustaining the development of ORS in vitro.

A Decoy Peptide Targeted to Protein Phosphatase 1 Attenuates Degradation of SERCA2a in Vascular Smooth Muscle Cells.

Neointimal growth in the injured vasculature is largely facilitated by the proliferation of vascular smooth muscle cells (VSMC), which associates with reduced sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) activity. The gene transfer-mediated restoration of the SERCA2a level thus attenuates neointimal growth and VSMC proliferation. We previously reported that a peptide targeted to protein phosphatase 1, ψPLB-SE, normalizes SERCA2a activity in cardiomyocytes. In this study, we found that ψPLB-SE attenuated neointimal growth in balloon-injured rat carotid arteries, and the proliferation and migration of VSMC cultured in high-serum media (synthetic conditions). In parallel, ψPLB-SE inhibited the degradation of SERCA2a in the injured carotid arteries and VSMC under synthetic conditions. The calpain inhibitor MDL28170 also attenuated SERCA2a degradation and VSMC proliferation under synthetic conditions, indicating that calpain degrades SERCA2a. The Ca2+ ionophore A23187 induced SERCA2a degradation in VSMC, which was blocked by either ψPLB-SE or MDL28170. Additionally, ψPLB-SE normalized the cytosolic Ca2+ level in VSMC that was increased by either A23187 or synthetic stimulation. Collectively, these data indicate that ψPLB-SE corrects the abnormal Ca2+ handling by activating SERCA2a, which further protects SERCA2a from calpain-dependent degradation in VSMC. We conclude that ψPLB-SE may form the basis of a therapeutic strategy for vascular proliferative disorders.

MP45-05 UROTHELIUM-SPECIFIC AND TEMPORALLY CONTROLLED BI-ALLELIC INACTIVATION OF BOTH PTEN AND P53 TRIGGERS BASAL-SUBTYPE MUSCLE-INVASIVE BLADDER CANCER

INTRODUCTION AND OBJECTIVES: PTEN is a tumor suppressor whose inhibitory activity towards phosphoinositide 3 kinase replies on membrane association, a process that is tightly regulated by electrostatic charges of PTEN tail domain. Precisely how and to what extent this domain affects PTEN’s activity remains unclear and is the subject of the present study. METHODS: Urothelial carcinoma cell lines without PTEN (UMUC3 and J82), or that expressing a mutated PTEN (T24), were transiently transfected with vector only, vector containing a full-length PTEN or vector containing a tailless PTEN, and their effects on cell proliferation, AKT activity, cell cycle and apoptosis were assessed under different culture conditions. To circumvent the extreme deleterious effects of the tailless PTEN, stable transfectants of UMUC3 were created that only expressed the tailless PTEN (or the full-length PTEN as a control) upon doxycycline exposure (e.g., stable transfection of CMV/rtTA and TRE-tailless PTEN (or TRE/full-length PTEN). The effects of their inducible expression were assessed in vitro and in xenograft mouse models. RESULTS: Introduction of full-length PTEN and tailless PTEN into PTEN-deficient UMUC3 and J82 cells inhibited proliferation at comparable levels in serum free and low serum (2%) media. However, in high serum (10%) medium, tailless PTEN was three times more inhibitory than full-length PTEN. This was due primarily to the phosphorylation of the serine/threonine clusters of the full-length PTEN’s tail domain, an effect absent in the tailless PTEN. Similar results were observed with transiently transfected T24 cells which expressed an endogenous mutant PTEN. UMUC3, J82 and T24 transfected with tailless PTEN could not sustain long-term culture because of its extreme deleterious effects, prompting us to establish doxycycline-inducible expression cell lines. UMUC3 inducibly expressing tailless PTEN exhibited significantly greater inhibition of proliferation, AKT phosphorylation, G1/S transition and significantly more apoptosis than UMUC3 inducibly expressing full-length PTEN. Finally, xenografted UMUC3 cells inducibly expressing full-length PTEN established tumors, albeit 30% smaller than the vector only controls. In contrast, those inducibly expressing tailless PTEN were devoid of any tumor in nude mice. CONCLUSIONS: Our in vitro and in vivo data are the first demonstration of the high potency of tailless PTEN as a tumor suppressor. They suggest that delivering tailless PTEN via viral vectors or other vehicles can be an effective approach to tackle advanced urothelial carcinomas with wild-type, mutated or deleted PTEN.

Culture and detection of primary cilia in endothelial cell models.

Background The primary cilium is a sensor of blood-induced forces in endothelial cells (ECs). Studies that have examined EC primary cilia have reported a wide range of cilia incidence (percentage of ciliated cells). We hypothesise that this variation is due to the diversity in culture conditions in which the cells are grown. We studied two EC types: human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells (HMEC-1s). Both cell types were grown in media containing foetal bovine serum (FBS) at high (20 % FBS and 10 % FBS for HUVECs and HMEC-1s, respectively) or low (2 % FBS) concentrations. Cells were then either fixed at confluence, serum-starved or grown post-confluence for 5 days in corresponding expansion media (cobblestone treatment). For each culture condition, we quantified cilia incidence and length.ResultsHUVEC ciliogenesis is dependent on serum concentration during the growth phase; low serum (2 % FBS) HUVECs were not ciliated, whereas high serum (20 % FBS) confluent HUVECs have a cilia incidence of 2.1 ± 2.2 % (median ± interquartile range). We report, for the first time, the presence of cilia in the HMEC-1 cell type. HMEC-1s have between 2.2 and 3.5 times greater cilia incidence than HUVECs (p < 0.001). HMEC-1s also have shorter cilia compared to HUVECs (3.0 ± 1.0 μm versus 5.1 ± 2.4 μm, at confluence, p = 0.003).Conclusions We demonstrate that FBS plays a role in determining the prevalence of cilia in HUVECs. In doing so, we highlight the importance of considering a commonly varied parameter (% FBS), in the experimental design. We recommend that future studies examining large blood vessel EC primary cilia use confluent HUVECs grown in high serum medium, as we found these cells to have a higher cilia incidence than low serum media HUVECs. For studies interested in microvasculature EC primary cilia, we recommend using cobblestone HMEC-1s grown in high serum medium, as these cells have a 19.5 ± 6.2 % cilia incidence.Electronic supplementary materialThe online version of this article (doi:10.1186/s13630-015-0020-2) contains supplementary material, which is available to authorized users.

Cell proliferation is a key determinant of the outcome of FOXO3a activation

The FOXO family of forkhead transcription factors have a pivotal role in determining cell fate in response to oxidative stress. FOXO activity can either promote cell survival or induce cell death. Increased FOXO-mediated cell death has been implicated in the pathogenesis of degenerative diseases affecting musculoskeletal tissues. The aim of this study was to determine the conditions under which one member of the FOXO family, FOXO3a, promotes cell survival as opposed to cell death. Treatment of primary human tenocytes with 1 pM hydrogen peroxide for 18 h resulted in increased protein levels of FOXO3a. In peroxide-treated cells cultured in low serum media, FOXO3a inhibited cell proliferation and protected against apoptosis. However in peroxide treated cells cultured in high serum media, cell proliferation was unchanged but level of apoptosis significantly increased. Similarly, in tenocytes transduced to over-express FOXO3a, cell proliferation was inhibited and level of apoptosis unchanged in cells cultured in low serum. However there was a robust increase in cell death in FOXO3a-expressing cells cultured in high serum. Inhibition of cell proliferation in either peroxide-treated or FOXO3a-expressing cells cultured in high serum protected against apoptosis induction. Conversely, addition of a Chk2 inhibitor to peroxide-treated or FOXO3a-expressing cells overrode the inhibitory effect of FOXO3a on cell proliferation and led to increased apoptosis in cells cultured in low serum. This study demonstrates that proliferating cells may be particularly susceptible to the apoptosis-inducing actions of FOXO3a. Inhibition of cell proliferation by FOXO3a may be a critical event in allowing the pro-survival rather than the pro-apoptotic activity of FOXO3a to prevail.

Nutrient deprivation increases vulnerability of endothelial cells to proinflammatory insults

Nutrient deprivation is a stimulus for oxidative stress and is an established method for induction of cell autophagy and apoptosis. The aims of this study were to identify conditions that evoke superoxide production in cultured human umbilical vein endothelial cells (HUVECs), determine the mechanism of action for this response, and examine whether the stimulus might facilitate the adhesion of human isolated neutrophils to the HUVECs. HUVECs were incubated in M199 medium under conditions of serum starvation (serum-free M199 medium), low serum (medium containing 2% fetal calf serum), and high serum (medium containing 20% fetal calf serum). HUVECs were also incubated under proinflammatory conditions, in medium supplemented with 50ng/ml tumor necrosis factor-α (TNF-α) or neutrophils preactivated with 10nM phorbol 12-myristate 13-acetate (PMA). Superoxide production was increased fourfold in serum-starved HUVECs compared to cells incubated in 20% medium, and this was reduced by inhibitors of the mitochondrial electron transport chain and mitochondrial Ca2+ uniporter. Superoxide production was 23.6% higher in HUVECs incubated with TNF-α in 2% medium compared to 2% medium alone, but unchanged with TNF-α in 20% medium. PMA-activated neutrophils adhered to morphologically aberrant HUVECs, which were mainly evident under the low-serum condition. The findings show a role of mitochondrial enzymes in superoxide production in response to nutrient deprivation and suggest that proinflammatory responses in HUVECs become manifest when HUVECs are in an already-compromised state.

The effect of serum in culture on RNAi efficacy through modulation of polyplexes size

Serum in the culture medium is one crucial factor that compromises RNAi efficiency of non-viral vectors. However, mechanistic roles of serum in siRNA delivery remain unknown. In this work, we took one cationic polymer, pullulan chemically modified by spermine (termed as pullulan-spermine, Ps), as a siRNA carrier model to investigate the effects of serum on key steps in siRNA delivery including formation of Ps and siRNA polyplexes (Ps-siRNA), cellular uptake, lysosomal escape, and cytotoxicity. We demonstrate that low serum concentration (1.25% and 2.5%) in culture medium results in large particles of Ps-siRNA, while high serum concentration (10%–40%) leads to small particles of Ps-siRNA. The larger particles initiated the internalization of siRNA more effectively in comparison to the smaller ones. The engulfed Ps-siRNA particles mainly locate in lysosomes. The large particles exhibited stronger abilities of destabilizing lysosomes than that of the small particles as large Ps-siRNA particles contain more amines and subsequently elicit a stronger proton sponge effect which results in more effective lysosomal escape of siRNA. Despite the lower RNAi efficiency, the small particle of Ps-siRNA in the high serum medium generates much lower cytotoxicity. These findings explain why serum significantly affects RNAi and also propose a strategy for improving RNAi efficiency and safety by modulating serum concentration and enhancing lysosomal destabilization.

CARP Plays a Key Role in Cellular Growth Under Hypertrophic Stimuli in Neonatal Rat Ventricular Myocytes

Cardiac hypertrophy is a growth process that occurs in response to stress stimuli or injury, and leads to the induction of several pathways to alter gene expression. Under hypertrophic stimuli, sarcomeric structure is disrupted, both as a consequence of gene expression and local changes in sarcomeric proteins. Cardiac-restricted ankyrin repeat protein (CARP) is one such protein that function both in cardiac sarcomeres and at the transcriptional level. We postulate that due to this dual nature, CARP plays a key role in maintaining the cardiac sarcomere. GATA4 is another protein detected in cardiomyocytes as important in hypertrophy, as it is activated by hypertrophic stimuli, and directly binds to DNA to alter gene expression. Results of GATA4 activation over time were inconclusive; however, the role of CARP in mediating hypertrophic growth in cardiomyocytes was clearly demonstrated. In this study, Neonatal Rat Ventricular Myocytes were used as a model to detect changes over time in CARP and GATA4 under hypertrophic stimulation by phenylephrine and high serum media. Results were detected by analysis of immunoblotting. The specific role that CARP plays in mediating cellular growth under hypertrophic stimuli was studied through immunofluorescence, which demonstrated that cardiomyocyte growth with hypertrophic stimulation was significantly blunted when NRVMs were co-treated with CARP siRNA. These data suggest that CARP plays an important role in the hypertrophic response in cardiomyocytes.

CaMKII Autonomy Is Substrate-dependent and Further Stimulated by Ca2+/Calmodulin

A hallmark feature of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) regulation is the generation of Ca(2+)-independent autonomous activity by Thr-286 autophosphorylation. CaMKII autonomy has been regarded a form of molecular memory and is indeed important in neuronal plasticity and learning/memory. Thr-286-phosphorylated CaMKII is thought to be essentially fully active ( approximately 70-100%), implicating that it is no longer regulated and that its dramatically increased Ca(2+)/CaM affinity is of minor functional importance. However, this study shows that autonomy greater than 15-25% was the exception, not the rule, and required a special mechanism (T-site binding; by the T-substrates AC2 or NR2B). Autonomous activity toward regular R-substrates (including tyrosine hydroxylase and GluR1) was significantly further stimulated by Ca(2+)/CaM, both in vitro and within cells. Altered K(m) and V(max) made autonomy also substrate- (and ATP) concentration-dependent, but only over a narrow range, with remarkable stability at physiological concentrations. Such regulation still allows molecular memory of previous Ca(2+) signals, but prevents complete uncoupling from subsequent cellular stimulation.

Measurements of Movement and Diffusion Coefficients of Single Cells on Polymeric Surface from Image Analysis

Time-lapse digital images were taken every 30 s of PC12 cells cultured on polystyrene dishes, collagen-coated dishes and poly(L-lysine)-coated dishes in high-serum medium, low-serum medium and neurite outgrowth factor (NGF)-containing medium to investigate their diffusion coefficients (i.e., self-diffusion coefficients), D and specific movement (i.e., specific frequent movement) using image analysis and Fast Fourier Transform (FFT) analysis. D for these cells was found to fluctuate as a function of time, D varying between 0 and 0.08 μm2/s. The trend observed upon examination of average D values was: D in high-serum medium ≥ D in low-serum medium ≥ D in NGF-containing medium. Image analysis and FFT analysis of single cells cultured on polymeric dishes in these three media did not have any specific frequency of cell movement between 0 and 0.0167 Hz. The high diffusion coefficient and high amplitude of power spectra of PC12 cells in high-serum medium might be attributed to the high energy necessary for their continual suppression of the mitogen-activated protein kinase (MAPK) cascade and for them to maintain their undifferentiated state.

Activity-dependent Regulation of h Channel Distribution in Hippocampal CA1 Pyramidal Neurons

The hyperpolarization-activated cation current, I(h), plays an important role in regulating intrinsic neuronal excitability in the brain. In hippocampal pyramidal neurons, I(h) is mediated by h channels comprised primarily of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel subunits, HCN1 and HCN2. Pyramidal neuron h channels within hippocampal area CA1 are remarkably enriched in distal apical dendrites, and this unique distribution pattern is critical for regulating dendritic excitability. We utilized biochemical and immunohistochemical approaches in organotypic slice cultures to explore factors that control h channel localization in dendrites. We found that distal dendritic enrichment of HCN1 is first detectable at postnatal day 13, reaching maximal enrichment by the 3rd postnatal week. Interestingly we found that an intact entorhinal cortex, which projects to distal dendrites of CA1 but not area CA3, is critical for the establishment and maintenance of distal dendritic enrichment of HCN1. Moreover blockade of excitatory neurotransmission using tetrodotoxin, 6-cyano-7-nitroquinoxaline-2,3-dione, or 2-aminophosphonovalerate redistributed HCN1 evenly throughout the dendrite without significant changes in protein expression levels. Inhibition of calcium/calmodulin-dependent protein kinase II activity, but not p38 MAPK, also redistributed HCN1 in CA1 pyramidal neurons. We conclude that activation of ionotropic glutamate receptors by excitatory temporoammonic pathway projections from the entorhinal cortex establishes and maintains the distribution pattern of HCN1 in CA1 pyramidal neuron dendrites by activating calcium/calmodulin-dependent protein kinase II-mediated downstream signals.

UltraSensitive Human Cytokine 10-Plex for the Luminex xMAP™ Platform (B200)

Abstract Profiling low concentrations of Th1/Th2 and proinflammatory cytokines can yield valuable information about immune system status in clinical and subclinical disease states. To facilitate cytokine profile analysis, Invitrogen has recently developed an UltraSensitive Cytokine 10-Plex Assay for the Luminex xMAP™ Platform. Analytes include human GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, and TNF-α. This assay system is validated for high content, high sensitivity serum, plasma, and tissue culture medium applications. Through careful choice of antibody pairs and use of proprietary buffer formulations, we are able to achieve an improvement in sensitivity of approximately 10-fold compared to our conventional cytokine assays for the Luminex xMAP™ platform. Sensitivities for all 10 analytes are 1 picogram per milliliter or less. Other performance characteristics, including assay precision, recovery, linearity of dilution, and calibration to NIBSC reference preparations, are similar to our conventional assays.

Dietary aflatoxin exposure and impaired growth in young children from Benin and Togo: cross sectional study

Profiling low concentrations of Th1/Th2 and proinflammatory cytokines can yield valuable information about immune system status in clinical and subclinical disease states. To facilitate cytokine profile analysis, Invitrogen has recently...

Extension of Sp2/0 hybridoma cell viability through interleukin-6 supplementation

Sp2/0 hybridoma cells die principally by apoptosis in batch culture. We have found that cultures of the Sp2/0 hybridoma exhibit increased viability in response to interleukin 6 (IL-6) supplementation relative to control cultures during serum shiftdown experiments. When shifted from a medium containing 10% fetal bovine serum (FBS) to a medium with 1% FBS, IL-6 supplemented cultures displayed viabilities and viable cell densities similar to control cultures containing 10% FBS. The degree of the survival response induced varied in accordance with the severity of the shiftdown, as cells resuspended in a high serum medium showed little observable enhancement in viability. The extension in culture viability was not accompanied by an observable decrease in growth relative to control cultures, indicating that the effect was not a consequence of growth inhibition. These results suggest the existence of serum components with behavior functionally similar to IL-6, with respect to enhancing cell survival, and that under certain experimental conditions IL-6 serves as a survival factor. In contrast to the extended viability displayed by cultures supplemented with IL-6, Sp2/0 cultures transfected with IL-6 cDNA expression vectors displayed a growth inhibitory response relative to control cultures. This inhibitory response was characterized by an extended lag phase following inoculation, and a decrease in batch culture cell yield. The depression in cell yield varied with serum concentration, with the largest depression occurring at high serum concentrations. We conclude that interactions between components in serum, presumably growth factors, and cytokines play an important role in altering the behavior of industrially relevant cell lines in culture. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 439-446, 1997.

Stimulation of cell proliferation and inhibition of gap junctional intercellular communication by linoleic acid

The effect of linoleic acid (LA) on gap-junction permeability, connexin 43 mRNA level, protein level, and phosphorylation, and the numbers of gap-junctional membrane plaques were studied in the rat liver epithelial cell line WB-F344 to determine whether changes in these parameters correlated with the enhanced cell growth and the inhibition of gap-junction function. When cultured in a medium with low serum (1%), these cells exhibited a slower growth rate than in the high serum medium (7%). Addition of linoleic acid (0.01–3 mg/ml) to the low serum medium increased the growth rate and inhibited gap junctional intercellular communication (GJIC) in a dose-dependent manner. In a comparison of short-term and long-term treatments with LA, GJIC in short-term treated (1 h) WB cells was inhibited at 3 mg/ml LA but readily recovered by washing and removing LA from cells, whereas GJIC in long-term treated (6 days) WB cells did not recover by washing and removing LA from WB cells. Western blot analysis of connexin 43 showed that a short-term incubation with linoleic acid increased the relative amount of unphosphorylated connexin 43 protein, but a long-term incubation with linoleic acid decreased the amount of unphosphorylated connexin 43 protein and increased the relative amount of hyperphosphorylated connexin 43 protein. Connexin 43 and p53 mRNA levels decreased in a time- and dose-dependent manner in linoleic acid-treated cells. These results suggest that growth stimulation and gap junctional intercellular communication inhibition of rat liver epithelial cells by linoleic acid may be mediated in part through modulation of p53 expression and function.

The expression of amyloid beta protein precursor protects nerve cells from β-amyloid and glutamate toxicity and alters their interaction with the extracellular matrix

The biological function of amyloid β protein precursor (ABPP) in nerve cells is examined by transfecting it into the B103 clonal nerve cell line which makes no ABPP and asking if a variety of biological activities are altered. Although ABPP is expressed by the transfected cells at high levels, there are no detectable differences between the ABPP negative control clones and the clones expressing ABPP with respect to growth rate in high serum medium, dibutyryl-cyclic AMP-induced differentiation, or morphology on plastic culture dishes. There are, however, significant differences in a number of properties between the two groups of cell lines. Cells expressing ABPP adhere more rapidly and have an enhanced rate of neurite outgrowth on collagen substrata. They also grow more rapidly in low serum medium. Finally, the expression of ABPP weakly but significantly protects the nerve cells from amyloid β protein and glutamate toxicity.