Mixed Cell Cultures Research Articles

Whole Genome Functional Analysis of Laminar Tissue From Horses With Laminitis Induced by Prolonged Euglycemic-Hyperinsulinemic Clamp

s Vol 30, No 2 (2010) 107 CHO administration or upon reaching Obel grade 3 lameness whichever occurred first. Horses in the treatment group received 8.5 mg/kg PTX in 1 L saline IV over 30 minutes beginning 12 hours prior to CHO administration, then every 12 hours thereafter until the completion of the study. MMP activity in the digital plasma will be evaluated using gelatin zymography. PRELIMINARY RESULTS All control horses (5/5) developed Obel grade 3 lameness, four of them within 32 hours post-CHO. However, only one PTX horse reached Obel grade 3. Of the remaining four PTX horses, two never became lame, one reached Obel grade 2 and then returned to almost complete soundness by 60 hours, and one fluctuated between Obel grades 1 and 2 during the entire study. MMP analysis is being performed at this time. DISCUSSION The horses appear to have responded favorably to PTX treatment with significantly decreased lameness. This response to PTX could result from 1) MMP inhibition, 2) anti-inflammatory effects of PTX, or 3) individual response to CHO. CLINICAL RELEVANCE If future data confirms our preliminary findings, PTX could be used as a preventative/treatment for cases of laminitis associated with carbohydrate overload. CONCLUSION PTX appears to decrease lameness associated with CHOinduced laminitis. Isolation of Equine Hoof Epidermal Cells for in Vitro Studies Hannah Galantino-Homer, Susan Megee, and Makoto Senoo, Laminitis Institute, Department of Clinical Studies/New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA, Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA TAKE HOME MESSAGE We present a method for aseptic retrieval of equine hoof epidermal cells. INTRODUCTION Disruption of basal epidermal cell-basement membrane adhesion is a key event during the developmental and acute phases of laminitis and disorganized proliferation of basal cells may contribute to the pathology of chronic laminitis. The isolation of these cells would facilitate in vitro studies of normal and disrupted basal cell-extracellular matrix adhesion and proliferation and could provide basal epidermal progenitor cells (BEPCs) for autografting following hoof wall resection. MATERIALS AND METHODS Aseptic surgical techniques are used to remove a 5 cm by 4 cm section of the dorsal hoof wall of equine cadavers up to 3 hr following euthanasia. Coronary and lamellar epidermal tissue and underlying dermal tissue are dissected by scalpel and washed in DPBS containing antibiotic/antimycotic and held in DMEM, 10% Fetal Calf Sera and antibiotic/antimycotic. Mechanical disruption by dicing, scraping, and electric homogenizer in combination with enzymatic digestion is used to generate a single cell suspension of mixed epidermal and dermal cells that are then frozen or used immediately for in vitro studies. RESULTS Mixed cells and selective culture have been used successfully in a preliminary experiment to generate BEPC holoclones. Non-selective in vitro culture of mixed cells results in non-uniform cell proliferation and differentiation. DISCUSSION AND CLINICAL RELEVANCE Selection of BEPCs is essential for repeatable culture of uniform populations of basal cells for laminitis pathophysiology studies as well as for successful therapeutic epidermal tissue regeneration. CONCLUSION Equine hoof BEPC selective culture from isolated mixed epidermal and dermal cells is currently under development. Chemical and Biological Properties of Aqueous Black Walnut Extracts That Contribute to the Pathogenesis of Laminitis D.J. Hurley, J.N. Moore, K.A. Hurley, L.J. Berghaus, K.L. Galland, and R.S.K. Majerle, Food Animal Health and Management Program, Department of Population Health, College of Veterinary Medicine, the University of Georgia, Athens, GA, Department of Large Animal Medicine, College of Veterinary Medicine, the University of Georgia, Athens, GA, Department of Chemistry, Hamline University, St. Paul, MN

Isolation of Equine Hoof Epidermal Cells for in Vitro Studies

Isolation of Equine Hoof Epidermal Cells for in Vitro Studies

Transcriptomic Analysis Identifies Phosphatases as Novel Targets for Adenotonsillar Hypertrophy of Pediatric Obstructive Sleep Apnea

Obstructive sleep apnea (OSA) is a highly prevalent disorder in children, in which enlarged adenotonsillar tissues (AT) play a major pathophysiologic role. Mechanisms leading to the proliferation and hypertrophy of AT in children who subsequently develop OSA remain unknown, and surgical extirpation of AT is associated with potential morbidity and mortality. We hypothesized that a computationally based analysis of gene expression in tonsils from children with OSA and children with recurrent tonsillitis without OSA can identify putative mechanistic pathways associated with tonsillar proliferation and hypertrophy in OSA. Palatine tonsils from children with either polysomnographically documented OSA or recurrent infectious tonsillitis were subjected to whole-genome microarray and functional enrichment analyses followed by significance score ranking based on gene interaction networks. The latter enabled identification and confirmation of a candidate list of tonsil-proliferative genes in OSA. In vitro studies using a mixed tonsil cell culture system targeting one of these candidates, phosphoserine phosphatase, revealed that it was more abundantly expressed in tonsils of children with OSA, and that pharmacological inhibition of phosphoserine phosphatase led to marked reductions in T- and B-lymphocyte cell proliferation and increased apoptosis. A systems biology approach revealed a restricted set of candidate genes potentially underlying the heightened proliferative properties of AT in children with OSA. Furthermore, functional studies confirm a novel role for protein phosphatases in AT hypertrophy, and may provide a promising strategy for discovery of novel, nonsurgical therapeutic targets in pediatric OSA.

Lipid-mediated delivery of RNA is more efficient than delivery of DNA in non-dividing cells

The design of appropriate gene delivery systems is essential for the successful application of gene therapy to clinical medicine. Cationic lipid-mediated delivery is a viable alternative to viral vector-mediated gene delivery in applications where transient gene expression is desirable. However, cationic lipid-mediated delivery of DNA to post-mitotic cells such as neurons is often reported to be of low efficiency, due to the presumed inability of the DNA to translocate to the nucleus. Lipid-mediated delivery of RNA is an attractive alternative to non-viral DNA delivery in some clinical applications, because transit across the nuclear membrane is not necessary. Here we report a comparative investigation of cationic lipid-mediated delivery of RNA versus DNA vectors encoding the reporter gene green fluorescent protein (GFP) in Chinese Hamster Ovary (CHO) and NIH3T3 cells following chemical inhibition of proliferation, and in primary mixed neuronal cell cultures. Using optimized formulations and transfection procedures, we assess gene expression by flow cytometry to specifically address some of the advantages and disadvantages of lipid-mediated RNA and DNA gene transfer. Despite inhibition of cell proliferation, over 45% of CHO cells express GFP after lipid-mediated transfection with RNA vectors. Transfection efficiency of DNA encoding GFP in proliferation-inhibited CHO cells was less than 5%. Detectable expression after RNA transfection occurs at least 3 h earlier than after DNA transfection, but DNA transfection eventually produces a mean level of per cell GFP expression (as assayed by flow cytometry) that is higher than after RNA transfection. Transfection of proliferation-inhibited NIH3T3 cells and primary mixed neuronal cultures produced similar results, with RNA encoded GFP expression in 2–4 times the number of cells as after DNA encoded GFP expression. These results demonstrate the increased efficiency of RNA transfection relative to DNA transfection in non-dividing cells. We used firefly luciferase encoded by RNA and DNA vectors to investigate the time course of gene expression after delivery of RNA or DNA to primary neuronal cortical cells. Delivery of mRNA resulted in rapid onset (within 1 h) of luciferase expression after transfection, a peak in expression 5–7 h after transfection, and a return to baseline within 12 h after transfection. After DNA delivery significant luciferase activity did not appear until 7 h after transfection, but peak luciferase expression was always at least one order of magnitude higher than after RNA delivery. The peak expression after luciferase-expressing DNA delivery occurred 36–48 h after transfection and remained at a significant level for at least one week before dropping to baseline. This observation is consistent with our in vivo delivery results, which are shown as well. RNA delivery may therefore be more suitable for short-term transient gene expression due to rapid onset, shorter duration of expression and greater efficiency, particularly in non-dividing cells. Higher mean levels of expression per cell obtained following DNA delivery and the longer duration of expression confirm a continuing role for DNA gene delivery in clinical applications that require longer term transient gene expression.

Neospora caninum: Early immune response of rat mixed glial cultures after tachyzoites infection

Neospora caninum causes neurologic disease in dogs and abortion in cattle. Little is known about the immune response of the CNS against this protozoan. The aim of this study was to evaluate production of IL-6, IL-10, TNF-α, IFN-γ, and NO in rat mixed glial cell cultures infected by N. caninum. IFN-γ was not observed. The mean cytokine released after 24 and 72 h of infection were 3.8 ± 0.6 and 3.7 ± 0.6 pg TNF-α/mg protein and 2.7 ± 0.69 and 4.1 ± 0.64 pg IL-10/mg protein, respectively, and more than 8.0 pg IL-6/mg protein for both time points. NO levels increased 24 h post-infection (2.3 ± 0.8 pg/mg protein) until 72 h (4.2 ± 1.1 pg/mg protein) and the number of tachyzoites reduced with the time. Our results show high levels of regulatory cytokines that may suppress the harmful effects of IFN-γ; high levels of TNF-α and NO may represent an effective response by infected glial cells against N. caninum.

A Mixed Cell Culture Model for Assessment of Proliferation in Tonsillar Tissues From Children with Obstructive Sleep Apnea or Recurrent Tonsillitis

A Mixed Cell Culture Model for Assessment of Proliferation in Tonsillar Tissues From Children with Obstructive Sleep Apnea or Recurrent Tonsillitis

The role of glial cells in influencing neurite extension by dorsal root ganglion cells

When pretreated with pertussis toxin (PTX), the neurites of adult rat dorsal root ganglion (DRG) cells in mixed cell cultures retract over a period of 2 h following the initial stimulus of removal from the cell culture incubator for brief periods of observation. The purpose of this investigation was to determine whether this PTX-dependent response was specific to any one of the three subpopulations of DRG neurons. However, no neurite retraction response was observed in neuron-enriched populations of cells, or in cultures enriched in isolectin B4 (IB4)-positive neurons or in IB4-negative neurons. But, the addition of non-neuronal cells, and/or medium conditioned by non-neuronal cells, was sufficient to restore the PTX-dependent neurite retraction response, but only in large diameter IB4-negative neurons. In conclusion, we have identified a regulatory response, mediated by Gi/o-proteins, which prevents retraction of neurites in large diameter IB4-negative cells of adult rat DRG. The non-neuronal cells of adult rat DRG constitutively release factor/s that can stimulate neurite retraction of a subset of isolated DRG neurons, but this property of non-neuronal cells is only observed when the Gi/o-proteins of large diameter IB4-negative cells are inhibited.

H19 RNA downregulation stimulated melanogenesis in melasma

A variety of factors, including ultraviolet (UV) exposure, have been implicated in the pathogenesis of melasma. However, UV-induced hyperpigmentation usually recovers spontaneously, whereas melasma does not. Recently, we detected downregulation of the H19 gene on microarray analysis of hyperpigmented and normally pigmented skin from patients with melasma, and identified significant clinical correlations. The H19 downregulation was not accompanied by a reciprocal change of the imprinted gene, insulin-like growth factor II. Moreover, methylation pattern of the H19 promoter region in maternal ICR was variable. The H19 knockdown in melanocyte monoculture did not result in obvious tyrosinase overexpression, whereas the knockdown in a mixed cell culture system, composed of H19 siRNA transfected normal human keratinocytes and non-transfected normal human melanocytes, did induce not only a tyrosinase overexpression but also an increase of melanosome transfer. Estrogen treatment of the H19 RNA knockdown in the mixed cell culture was more than an additive effect on the tyrosinase overexpression, whereas UV irradiation was not. These findings suggest that downregulation of H19 and a sufficient dose of estrogen might be involved in the development of melasma.

Therapeutic efficacy of VP_(22) and cytosine deaminase gene modified rat neural stem cell in treatment of C6 glioma in vitro

Objective To explore the therapeutic efficacy of rat neural stem cells genetically engineered by VP_(22) and cytosine deaminase (CD) gene in the treatment of C6 glioma cells in vitro. Method The encoding CD gene fragment was got from pAdTrack-CMV-CD by polymerase chain reaction (PCR),and cloned into pHIV-EGFP and pHIV-VP_(22)-EGFP to yield pHIV-CD-EGFP and pHIV-VP_(22)-CD-EGFP, the constructors were confirmed by the restriction enzyme method and PCR. The three-plasmid system containing constructors of pHIV-EGFP plasmid, helper plasmid and envelope plasmid were co-transfected into 293T cells. The viral particles were collected, and infected rat neural stem cells(NSCs) which were cultured from pregnant SD rat brain. The expression of CD transcripts in NSCs/CD -EGFP and NSCs/VP_(22)-CD-EGFP were identified by RT-PCR. The NSCs transfected by lentivirus-EGFP, lcntivirus-CD-EGFP, lentivirus-VP_(22)-EGFP or lentivirus-VP_(22)-CD-EGFP were co-cultured with C6 glioma cells at a ratio of 1:1 respectively. 5-FC was added to the mixed cell cultures for 3days at the final concentration of 100 mg/L and cell viability was performed by MTT assay. Results The cloning sites of the encoding CD fragment in pHIV-EGFP and pHIV-VP_(22)-EGFP were confirmed by the restriction enzyme method and PCR. In the presence of 5-FC, the cell survival rates of C6 glioma cells co-cultrued with the rat NSCs transfected by lentivirus-EGFP, lentivirus-CD-EGFP, lentivirus-VP_(22)-EGFP or lentivirus-VP_(22)-CD-EGFP were (95.57±4.83)%, (49.96±7.19)%, (94.25±4.32)% and (28.06±6.26)% respectively, showed significant growth inhibition (P<0.01) in the presence of CD gene,and the presence of VP_(22)-CD gene were much more sensitive to 5-FC than the presence of CD gene only(P<0.01). Conclusions VP_(22) increases the cell-killing effect of neural stem cells modified by CD gene in C6 glioma in vitro. Key words: Lentivirus vector; VP_(22) shuttle protein; Neural stem cells; C6 glioma cell

Interleukin-25 Expressed by Brain Capillary Endothelial Cells Maintains Blood-Brain Barrier Function in a Protein Kinase Cϵ-dependent Manner

Interleukin (IL)-25, a member of the IL-17 family of cytokines, is expressed in the brains of normal mice. However, the cellular source of IL-25 and its function in the brain remain to be elucidated. Here, we show that IL-25 plays an important role in preventing infiltration of the inflammatory cells into the central nervous system. Brain capillary endothelial cells (BCECs) express IL-25. However, it is down-regulated by inflammatory cytokines, including tumor necrosis factor (TNF)-alpha, IL-17, interferon-gamma, IL-1beta, and IL-6 in vitro, and is also reduced in active multiple sclerosis (MS) lesions and in the inflamed spinal cord of experimental autoimmune encephalomyelitis, an animal model of MS. Furthermore, IL-25 restores the reduced expression of tight junction proteins, occludin, junction adhesion molecule, and claudin-5, induced by TNF-alpha in BCECs and consequently repairs TNF-alpha-induced blood-brain barrier (BBB) permeability. IL-25 induces protein kinase Cepsilon (PKCepsilon) phosphorylation, and up-regulation of claudin-5 is suppressed by PKCepsilon inhibitor peptide in the IL-25-stimulated BCECs. These results suggest that IL-25 is produced by BCECs and protects against inflammatory cytokine-induced excessive BBB collapse through a PKCepsilon-dependent pathway. These novel functions of IL-25 in maintaining BBB integrity may help us understand the pathophysiology of inflammatory brain diseases such as MS.

Microglia Activated with the Toll-Like Receptor 9 Ligand CpG Attenuate Oligomeric Amyloid β Neurotoxicity in in Vitro and in Vivo Models of Alzheimer’s Disease

Soluble oligomeric amyloid beta (oAbeta) 1-42 causes synaptic dysfunction and neuronal injury in Alzheimer's disease (AD). Although accumulation of microglia around senile plaques is a hallmark of AD pathology, the role of microglia in oAbeta1-42 neurotoxicity is not fully understood. Here, we showed that oAbeta but not fibrillar Abeta was neurotoxic, and microglia activated with unmethylated DNA CpG motif (CpG), a ligand for Toll-like receptor 9, attenuated oAbeta1-42 neurotoxicity in primary neuron-microglia co-cultures. CpG enhanced microglial clearance of oAbeta1-42 and induced higher levels of the antioxidant enzyme heme oxygenase-1 in microglia without producing neurotoxic molecules such as nitric oxide and glutamate. Among subclasses of CpGs, class B and class C activated microglia to promote neuroprotection. Moreover, intracerebroventricular administration of CpG ameliorated both the cognitive impairments induced by oAbeta1-42 and the impairment of associative learning in Tg2576 mouse model of AD. We propose that CpG may be an effective therapeutic strategy for limiting oAbeta1-42 neurotoxicity in AD.

The human alloreactive CD4+ T-cell repertoire is biased to a Th17 response and the frequency is inversely related to the number of HLA class II mismatches

AbstractEstimates of precursor frequency and assessment of functional characteristics of alloreactive CD4+ T cells are all biased by the need for long-term culture. In this study, direct visualization of human alloreactive CD4+ T cells on the single-cell level was achieved using cell surface expression of CD154 as a tool for identification. The average frequency of alloreactive CD154+CD4+ T cells among peripheral blood CD4+ T cells was 0.1%, with half of the cells displaying a naive phenotype. The proliferation capacity and expression of cytokines after allogeneic stimulation resided in these CD154+CD4+ T cells. The repertoire of alloreactive CD4+ T cells was biased to a Th17 response, and on average 24% of alloreactive CD154+CD4+ memory T cells produced interleukin-17 (IL-17) after polyclonal stimulation. Unexpectedly, mixed cell cultures from human leukocyte antigen (HLA)–identical donors also generated alloreactive CD154+CD4+ T cells and yielded the highest frequency compared with HLA-nonidentical combinations. Therefore, reactivity to minor histocompatibility antigens between HLA-identical subjects appears to be relatively common. Alloreactive HLA-identical T cells did not proliferate or express cytokines, but were driven to proliferation in the presence of exogenous IL-2.

Ibuprofen for neuroprotection after cerebral ischemia

Ibuprofen has been shown to reduce cerebral ischemic injury, such as may occur after deep hypothermic circulatory arrest. We investigated whether ibuprofen has direct protective effects against excitotoxic neuronal injury, as may be seen after cerebral ischemia, by using a cell culture model. Mixed cortical cultures containing neuronal and glial cells were prepared from fetal mice at 13 to 15 days gestation, plated on a layer of confluent astrocytes from 1- to 3-day-old postnatal pups. Near-pure neuronal cultures containing less than 5% astrocytes were obtained from mice of the same gestational stage. Slowly triggered excitotoxic injury was induced at 37 degrees C by 24-hour exposure to 12.5 micromol/L N-methyl-D-aspartate or 50 micromol/L kainate. Neuronal death was quantified by release of lactate dehydrogenase from damaged cells. Data were analyzed using 1-way analysis of variance with Tukey post hoc multiple comparisons. In mixed cultures, ibuprofen concentrations of 25 microg/mL, 50 microg/mL, and 100 microg/mL all significantly reduced N-methyl-D-aspartate-induced neuronal cell death from 74.5% to 56.1%, 38.7%, and 12.3%, respectively, revealing a strong dose response (P < .001). In near-pure cultures, ibuprofen at a concentration of 25 microg/mL failed to protect neurons, indicating that the neuroprotective effects of ibuprofen require interaction with glial cells. Furthermore, ibuprofen at 100 microg/mL was not protective against neuronal cell death induced by kainate excitotoxicity in near-pure culture but was effective in mixed cultures. Ibuprofen provides neuroprotection through glial cells against excitotoxic neuronal injury caused by glutamatergic excitotoxicity after cerebral ischemia as demonstrated by reduced neuronal cell death in mixed cell cultures. Further studies are needed to evaluate the potential of ibuprofen to reduce neurologic injury in patients experiencing an hypoxic/ischemic insult.

Increased Cellular Proliferation and Inflammatory Cytokines in Tonsils Derived From Children With Obstructive Sleep Apnea

Adenotonsillar hypertrophy is the major pathophysiological mechanism underlying obstructive sleep apnea (OSA) and recurrent tonsillitis (RI) in children. The increased expression of various mediators of the inflammatory response in tonsils of patients with OSA prompted our hypothesis that the enhanced local and systemic inflammation in children with OSA would promote tonsillar proliferation. Mixed cell cultures from tonsils recovered during adenotonsillectomy in children with OSA and RI were established, and proliferative rates were assessed. Cells were also cultured to determine the levels of proinflammatory cytokines and antioxidant protein levels and mRNA expression. Global cell proliferative rates from OSA tonsils were significantly higher than RI (p < 0.01), with CD3, CD4, and CD8 cell proliferation being higher in OSA (p < 0.05). Moreover, proinflammatory cytokines, such as TNF-alpha, IL-6, and IL-1alpha, were highly expressed in OSA-derived tonsils. Furthermore, thioredoxin (TRX), an antioxidant protein, was also highly expressed in OSA tonsils at the mRNA and protein levels (p < 0.01). Thus, T cells are in a highly proliferative state in the tonsils of children with OSA and are associated with increased production of proinflammatory cytokines and TRX, when compared with children with RI.

Ketamine Inhibits Maturation of Bone Marrow-Derived Dendritic Cells and Priming of the Th1-Type Immune Response

Dendritic cells (DCs) play a key role as antigen-presenting cells and growing evidence suggests that DCs influence T-cell activation and regulate the polarity of the immune response. Ketamine has been reported to have immunomodulatory properties that affect immune cells, including macrophages and natural killer cells. However, the effect of ketamine on DCs has not been characterized. We examined the immunomodulation of DCs by ketamine. We used bone marrow-derived DCs induced by granulocyte-monocyte-colony stimulating factor and interleukin (IL)-4 from bone marrow and analyzed the expression of costimulatory molecules (CD40, CD80, and CD86), major histocompatibility complex class II molecules, and secretion of IL-12p40. Furthermore, we evaluated the immune response in mixed cell cultures of DCs and T cells and the contact hypersensitivity response in a whole animal. Ketamine suppressed the expression of CD40, CD80, and major histocompatibility complex class II molecules in DCs. DCs treated with ketamine also secreted less IL-12p40 and displayed greater endocytosis. In mixed cell cultures with CD4+ T cells and DCs, ketamine-treated DCs showed less propensity to stimulate the proliferation of CD4+ T cells and the secretion of interferon from CD4+ T cells. Furthermore, ketamine-treated DCs impaired the induction of a cell-mediated immune response. Our findings suggest that ketamine inhibits the functional maturation of DCs and interferes with DC induction of Th1 immunity in the whole animal. These novel findings provide new insight into the immunopharmacological role of ketamine.

Increase of intracellular Ca2+ by P2Y but not P2X receptors in cultured cortical multipolar neurons of the rat

AbstractThe expression and functionality of P2X/P2Y receptor subtypes in multipolar nonpyramidal neurons of mixed cortical cell cultures were investigated by means of immunocytochemistry and fura‐2 microfluorimetry. The morphological studies revealed that most of the neurons are immunoreactive for GABA and express a range of P2X/P2Y receptors, predominantly of the P2X2,4,6 and P2Y1,2 subtypes. P2X1 and P2X7 receptor immunoreactivity (IR) was found on thin axon‐like processes and presynaptic structures, respectively. Application of ATP caused a small concentration‐dependent increase in intracellular Ca2+ concentration ([Ca2+]i) in most investigated neurons, whereas only about the half of these cells responded to 2′,3′‐O‐(benzoyl‐4‐benzoyl)‐ATP (BzATP), ADPβS, 2MeSADP, or 2‐MeSATP and even fewer cells to UTP. In contrast, α,β‐meATP, UDP, and UDP‐glucose failed to produce any [Ca2+]i signaling. The response to ATP itself was inhibited by pyridoxal‐5′‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS), Reactive Blue 2, 2′‐deoxy‐N6‐methyl adenosine 3′,5′‐diphosphate (MRS2179), and suramin (300 μM) as well as by a cyclopiazonic acid‐induced depletion of intracellular Ca2+ stores. A Ca2+‐free external medium tended to decrease the ATP‐induced [Ca2+]i transients, although this action did not reach statistical significance. Various blockers of voltage‐sensitive Ca2+ channels and the gap junction inhibitor carbenoxolone did not interfere with the effect of ATP, whereas a combination of the ionotropic glutamate receptor antagonists D(–)‐2‐amino‐ 5‐phosphonopentanoic acid (AP5) and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione disodium (CNQX) decreased it. Cross‐desensitization experiments between ADPβS or UTP and ATP suggested that ATP acts on the one hand via P2Y1,2 receptors and on the other hand by additional signaling mechanisms. These mechanisms may involve the release of glutamate (which in consequence activates ionotropic glutamate receptors) and the entry of Ca2+ via store‐operated Ca2+ channels. Evidence for the presence of functional P2X receptors, in particular P2X7, remains elusive. J. Comp. Neurol. 516:343–359, 2009. © 2009 Wiley‐Liss, Inc.

Increase of intracellular Ca2+ by P2Y but not P2X receptors in cultured cortical multipolar neurons of the rat

AbstractThe expression and functionality of P2X/P2Y receptor subtypes in multipolar nonpyramidal neurons of mixed cortical cell cultures were investigated by means of immunocytochemistry and fura‐2 microfluorimetry. The morphological studies revealed that most of the neurons are immunoreactive for GABA and express a range of P2X/P2Y receptors, predominantly of the P2X2,4,6 and P2Y1,2 subtypes. P2X1 and P2X7 receptor immunoreactivity (IR) was found on thin axon‐like processes and presynaptic structures, respectively. Application of ATP caused a small concentration‐dependent increase in intracellular Ca2+ concentration ([Ca2+]i) in most investigated neurons, whereas only about the half of these cells responded to 2′,3′‐O‐(benzoyl‐4‐benzoyl)‐ATP (BzATP), ADPβS, 2MeSADP, or 2‐MeSATP and even fewer cells to UTP. In contrast, α,β‐meATP, UDP, and UDP‐glucose failed to produce any [Ca2+]i signaling. The response to ATP itself was inhibited by pyridoxal‐5′‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS), Reactive Blue 2, 2′‐deoxy‐N6‐methyl adenosine 3′,5′‐diphosphate (MRS2179), and suramin (300 μM) as well as by a cyclopiazonic acid‐induced depletion of intracellular Ca2+ stores. A Ca2+‐free external medium tended to decrease the ATP‐induced [Ca2+]i transients, although this action did not reach statistical significance. Various blockers of voltage‐sensitive Ca2+ channels and the gap junction inhibitor carbenoxolone did not interfere with the effect of ATP, whereas a combination of the ionotropic glutamate receptor antagonists D(–)‐2‐amino‐5‐phosphonopentanoic acid (AP5) and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione disodium (CNQX) decreased it. Cross‐desensitization experiments between ADPβS or UTP and ATP suggested that ATP acts on the one hand via P2Y1,2 receptors and on the other hand by additional signaling mechanisms. These mechanisms may involve the release of glutamate (which in consequence activates ionotropic glutamate receptors) and the entry of Ca2+ via store‐operated Ca2+ channels. Evidence for the presence of functional P2X receptors, in particular P2X7, remains elusive. J. Comp. Neurol. 516:343–359, 2009. © 2009 Wiley‐Liss, Inc.

Increase of intracellular Ca2+ by P2Y but not P2X receptors in cultured cortical multipolar neurons of the rat

The expression and functionality of P2X/P2Y receptor subtypes in multipolar nonpyramidal neurons of mixed cortical cell cultures were investigated by means of immunocytochemistry and fura-2 microfluorimetry. The morphological studies revealed that most of the neurons are immunoreactive for GABA and express a range of P2X/P2Y receptors, predominantly of the P2X(2,4,6) and P2Y(1,2) subtypes. P2X(1) and P2X(7) receptor immunoreactivity (IR) was found on thin axon-like processes and presynaptic structures, respectively. Application of ATP caused a small concentration-dependent increase in intracellular Ca2+ concentration ([Ca2+]i) in most investigated neurons, whereas only about the half of these cells responded to 2',3'-O-(benzoyl-4-benzoyl)-ATP (BzATP), ADPbetaS, 2MeSADP, or 2MeSATP and even fewer cells to UTP. In contrast, alpha,beta-meATP, UDP, and UDP-glucose failed to produce any [Ca2+]i signaling. The response to ATP itself was inhibited by pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), Reactive Blue 2, 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate (MRS2179), and suramin (300 microM) as well as by a cyclopiazonic acid-induced depletion of intracellular Ca2+ stores. A Ca2+-free external medium tended to decrease the ATP-induced [Ca2+]i transients, although this action did not reach statistical significance. Various blockers of voltage-sensitive Ca2+ channels and the gap junction inhibitor carbenoxolone did not interfere with the effect of ATP, whereas a combination of the ionotropic glutamate receptor antagonists D(-)-2-amino-5-phosphonopentanoic acid (AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) decreased it. Cross-desensitization experiments between ADPbetaS or UTP and ATP suggested that ATP acts on the one hand via P2Y(1,2) receptors and on the other hand by additional signaling mechanisms. These mechanisms may involve the release of glutamate (which in consequence activates ionotropic glutamate receptors) and the entry of Ca2+ via store-operated Ca2+ channels. Evidence for the presence of functional P2X receptors, in particular P2X(7), remains elusive.

Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation

Glypicans are heparan sulfate proteoglycans that are bound to the cell surface by glycosylphosphatidylinositol. While six members of the glypican family are known in mammals, our study focused on glypican 3 (GPC3). Loss-of-function mutations of GPC3 result in the Simpson-Golabi-Behmel syndrome, an X-linked disorder characterized by pre- and postnatal liver and other organ overgrowth. GPC3 is overexpressed in human hepatocellular carcinoma; however, its role in normal liver regeneration and hepatocyte proliferation is unknown. Here we investigated the role of GPC3 in hepatocyte proliferation. GPC3 mRNA and protein levels begin to increase 2 days after hepatectomy with peak expression levels by day 5. In hepatocyte cultures, GPC3 reaches a plateau when hepatocyte proliferation decreases. In vitro studies using Morpholino oligonucleotides showed that blocking GPC3 expression promoted hepatocyte growth. Yeast two-hybrid assays revealed that GPC3 interacts with CD81, a member of the tetraspanin family that is reported to be involved in hepatitis C virus infection and cell proliferation. We found that CD81 levels also increased 2 days after partial hepatectomy and toward the end of regeneration. Immunofluorescence showed that CD81 and GPC3 colocalize by 2 and 6 days after hepatectomy. Co-immunoprecipitation validated the interaction of GPC3 and CD81. Our results indicate that GPC3 may be a negative regulator of liver regeneration and hepatocyte proliferation, and that this regulation may involve CD81.

STIRRING FREE SURFACE FLOWS DUE TO HORIZONTAL CIRCULATORY OSCILLATION OF A PARTIALLY FILLED CONTAINER

Mixing in a partially filled beaker or conical flask using oscillator shaker tables is routinely used for cultivation of biological cells. The present study seeks to gain a better understanding of fluid flow inside a beaker on a shaker table and the fluid motion influence on the mixing of cells within the fluid medium used for their cultivation. The imposed oscillatory motion on the beaker induces free surface deformations, which may cause laminar and low shear flow environments thus uniformly mixing the fluid medium. The low shear and uniformly mixed cell culture medium are requirements necessary for optimum cellular growth. In this study, fluid flow due to periodic and horizontal circulatory oscillations at a beaker boundary has been analyzed both numerically and experimentally. A partially filled beaker was modeled as a circular cylinder, and the cell culture medium was modeled as an incompressible fluid with a free and deformable liquid interface. The three-dimensional numerical model that can resolve the free surface deformation was based on finite difference scheme based on the “Marker-and-Cell” method. The method was used to determine the free surface deformation, its coupling to the flow dynamics within the beaker, and the resulting stirring effects. To verify the numerical model and validate the results, a simple flow visualization experiment was performed using an ion laser sheet to optically section flow chamber and reveal the flow pattern. The flow patterns obtained using numerical simulations were similar to those obtained experimentally through flow visualization. This study provides useful information that can be used to optimize the operation of a shaker table necessary to mix uniformly the cell culture, ensuring cell growth. In addition, this model can be extended to study the mixing processes in any chemical reactions within a partially filled container subjected to oscillatory forces.