sq-23377 and Disease-Models--Animal

Ephedra herb (Mao) exerts potent anti-allergic effects. This study aimed to examine the underlying mechanisms of Mao on allergic inflammation using in vitro cultured mast cells (MCs) and an in vivo model of MC-dependent anaphylaxis.. Bone marrow-derived MCs (BMMCs) were presensitized with anti-2,4-dinitrophenol (DNP) immunoglobulin E (IgE) and challenged with antigens (Ag; DNP-human serum albumin). Degranulation responses and cell surface high-affinity receptor for IgE (FcεRI) expression were assessed with/without Mao treatment. Passive systemic anaphylaxis (PSA)-treated mice were administered Mao and the pathophysiological responses were evaluated.. Mao inhibited Ag-induced BMMC degranulation, but not polyclonal activation with phorbol 12-myristate 13-acetate (PMA) and ionomycin, indicating that Mao inhibits IgE-dependent activation of BMMCs. Mao-treated BMMCs exhibited significant reductions in expression of surface IgE and its receptor FcεRI. Analysis of subcellular localization revealed that Mao induces FcεRI internalization in BMMCs without degranulation. In the PSA mouse model, Mao administration prevented antigen-induced hypothermia. Mao administration significantly reduced cell surface expression of IgE-bound FcεRI on peritoneal MCs.. Mao induced FcεRI internalization in MCs, thereby inhibiting Ag-induced IgE-dependent degranulation. The inhibitory effects of Mao on MC degranulation may offer a novel therapeutic approach for allergic diseases.

Topics: Anaphylaxis; Animals; Anti-Allergic Agents; Cell Degranulation; Cells, Cultured; Disease Models, Animal; Ephedra; Female; Humans; Immunoglobulin E; Ionomycin; Mast Cells; Medicine, Kampo; Mice; Plant Extracts; Plant Stems; Primary Cell Culture; Signal Transduction; Tetradecanoylphorbol Acetate

Topics: Animals; Antigen-Presenting Cells; Antigens, CD; Cancer Vaccines; CD8-Positive T-Lymphocytes; Disease Models, Animal; Humans; Ionomycin; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocyte Depletion; Mice, Inbred NOD; Mice, SCID

ESSENTIALS: Neutrophil elastase (NE) plays a role in extracellular trap formation (NETosis) triggered by microbes. The contribution of NE was evaluated in mouse NETosis models of sterile inflammation and thrombosis. NE is not required for mouse neutrophil NET production in vitro with non-infectious stimuli. NE deficiency had no significant effect on thrombosis in the inferior vena cava stenosis model.. Neutrophil serine proteases have been implicated in coagulation and neutrophil extracellular trap (NET) formation. In human neutrophils, neutrophil elastase (NE) translocates to the nucleus during NETosis and cleaves histones, thus aiding in chromatin decondensation. NE(-/-) mice were shown not to release NETs in response to microbes. However, mouse studies evaluating the role of NE in NET formation in sterile inflammation and thrombosis are lacking.. We wished to establish if neutrophils from NE(-/-) mice have a defect in NETosis, similar to peptidylarginine deiminase 4 (PAD4(-/-)) mice, and how this might have an impact on venous thrombosis, a model where NETs are produced and are crucial to thrombus development.. We performed in vitro NET assays using neutrophils from wild-type (WT), NE(-/-), SerpinB1 (SB1)(-/-) and NE(-/-) SB1(-/-) mice. We compared WT and NE(-/-) animals using the inferior vena cava stenosis model of deep vein thrombosis (DVT).. Neutrophil elastase deficiency resulted in a small reduction in ionomycin-induced NET formation in vitro without affecting histone citrullination. However, NET production in response to phorbol 12-myristate 13-acetate or platelet activating factor was normal in neutrophils from two independent NE-deficient mouse lines, and in NE(-/-) SB1(-/-) as compared with SB1(-/-) neutrophils. NE deficiency or inhibition did not prevent NETosis in vivo or DVT outcome.. Neutrophil elastase is not required for NET formation in mice. NE(-/-) mice, which form pathological venous thrombi containing NETs, do not phenocopy PAD4(-/-) mice in in vitro NETosis assays or experimental venous thrombosis. Our study suggests that NET-targeted therapies need to be highly effective to have an impact on DVT.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Extracellular Traps; Genotype; Ionomycin; Leukocyte Elastase; Mice, Inbred C57BL; Mice, Knockout; Neutrophil Activation; Neutrophils; Phenotype; Tetradecanoylphorbol Acetate; Venous Thrombosis

Experimental autoimmune encephalomyelitis (EAE) is T-cell-dependent disease of the central nervous system (CNS) of mice. This model resembles multiple sclerosis (MS) in many aspects. Therapies that focus in the modulation of the immune response and cellular infiltration in the CNS present best effects in the clinics. Artesunate (Art) is a semi-synthetic sesquiterpene derivative from artemisinin and has been shown to reduce the clinical signs of autoimmune disease models through mechanisms not yet understood. In this study, we aimed to evaluate whether administration of Art would ameliorate EAE.. C57BL6 mice were immunized with MOG35-55 peptide to induce EAE. At the same time, Art treatment started (3 mg/kg/day via i.p.) for five consecutive days. We found that Art treatment reduced the clinical signs of EAE and that correlated with a reduced infiltration of cells in the CNS. Disease amelioration did not correlate with immunomodulation as recall responses, leukocyte subpopulations, and gene expression analysis were similar among treated and untreated mice. Ultimately, further analysis provided data indicating that a possible mechanism of action for Art is dependent on the cellular migration to the CNS.. Artesunate reduces the severity of EAE by inhibiting migration of pathogenic T cells to the CNS.

Topics: Analysis of Variance; Animals; Anti-Inflammatory Agents; Artemisinins; Artesunate; Brefeldin A; Cell Movement; Central Nervous System; Cytokines; Disease Models, Animal; DNA-Binding Proteins; Encephalomyelitis, Autoimmune, Experimental; Enzyme Inhibitors; Female; Flow Cytometry; Gene Expression Regulation; Ionomycin; Leukocytes; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Phorbol Esters

Perinatal hypoxic-ischaemic encephalopathy (HIE) occurs in 1-2 in every 1000 term infants and the devastating consequences range from cerebral palsy, epilepsy and neurological deficit to death. Cellular damage post insult occurs after a delay and is mediated by a secondary neural energy failure. AMP-activated protein kinase (AMPK) is a sensor of cellular stress resulting from ATP depletion and/or calcium dysregulation, hallmarks of the neuronal cell death observed after HIE. AMPK activation has been implicated in the models of adult ischaemic injury but, as yet, there have been no studies defining its role in neonatal asphyxia. Here, we find that in an in vivo model of neonatal hypoxia-ischaemic and in oxygen/glucose deprivation in neurons, there is pathological activation of the calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ)-AMPKα1 signalling pathway. Pharmacological inhibition of AMPK during the insult promotes neuronal survival but, conversely, inhibiting AMPK activity prior to the insult sensitizes neurons, exacerbating cell death. Our data have pathological relevance for neonatal HIE as prior sensitization such as exposure to bacterial infection (reported to reduce AMPK activity) produces a significant increase in injury. We show that in an in vivo model of neonatal hypoxia-ischaemic and in oxygen/glucose deprivation in neurons, there is a pathological activation of the CaMKKβ-AMPKα1 signalling pathway. Inhibiting AMPK during OGD promotes neuronal survival; conversely, inhibiting AMPK prior to OGD exacerbates cell death. Our data have clinical relevance as prior sensitization (e.g. exposure to bacterial infection reducing AMPK activity) increases injury. AMPK, AMP-activated protein kinase; HI, hypoxia-ischaemia; OGD, oxygen-glucose deprivation.

Topics: AMP-Activated Protein Kinases; Animals; Animals, Newborn; Benzimidazoles; Brain; Cell Death; Cells, Cultured; Disease Models, Animal; Enzyme Inhibitors; Gene Expression Regulation, Developmental; Glucose; Hypoxia; Hypoxia-Ischemia, Brain; Ionomycin; L-Lactate Dehydrogenase; Mice; Mice, Inbred C57BL; Naphthalimides; Neurons; Signal Transduction; Time Factors

Artificial oocyte activation to overcome failed fertilization after intracytoplasmic sperm injection (ICSI) in human oocytes typically employs Ca(2+) ionophores to produce a single cytosolic Ca(2+) increase. In contrast, recombinant phospholipase Czeta (PLCζ) causes Ca(2+) oscillations indistinguishable from those occurring during fertilization, but remains untested for its efficacy in a scenario of ICSI fertilization failure. Here, we compare PLCζ with other activation stimuli in a mouse model of failed oocyte activation after ICSI, in which heat-treated sperm are injected into mouse oocytes. We show that increasing periods of 56 °C exposure of sperm produces a progressive loss of Ca(2+) oscillations after ICSI. The decrease in Ca(2+) oscillations produces a reduction in oocyte activation and embryo development to the blastocyst stage. We treated such oocytes that failed to activate after ICSI either with Ca(2+) ionophore, or with Sr(2+) media which causes Ca(2+) oscillations, or we injected them with recombinant human PLCζ. All these treatments rescued oocyte activation, although Sr(2+) and PLCζ gave the highest rates of development to blastocyst. When recombinant PLCζ was given to oocytes previously injected with control sperm, they developed normally to the blastocyst stage at rates similar to that after control ICSI. The data suggest that recombinant human PLCζ protein is an efficient means of rescuing oocyte activation after ICSI failure and that it can be effectively used even if the sperm already contains endogenous Ca(2+) releasing activity.

Topics: Animals; Blastocyst; Calcium Signaling; Disease Models, Animal; Embryonic Development; Female; Fertilization; Hot Temperature; Humans; Infertility, Male; Ionomycin; Ionophores; Male; Mice; Mice, Inbred Strains; Oocytes; Phosphoinositide Phospholipase C; Recombinant Fusion Proteins; Sperm Injections, Intracytoplasmic; Strontium

Chronic spinal cord injury (SCI) induces immune depression in patients, which contributes to their higher risk of developing infections. While defects in humoral immunity have been reported, complications in T-cell immunity during the chronic phase of SCI have not yet been explored.. To assess the impact of chronic SCI on peripheral T-cell number and function we used a mouse model of severe spinal cord contusion at thoracic level T9 and performed flow cytometry analysis on the spleen for T-cell markers along with intracellular cytokine staining. Furthermore we identified alterations in sympathetic activity in the spleen of chronic SCI mice by measuring splenic levels of tyrosine hydroxylase (TH) and norepinephrine (NE). To gain insight into the neurogenic mechanism leading to T-cell dysfunction we performed in vitro NE stimulation of T-cells followed by flow cytometry analysis for T-cell exhaustion marker.. Chronic SCI impaired both CD4+ and CD8+ T-cell cytokine production. The observed T-cell dysfunction correlated with increased expression of programmed cell death 1 (PD-1) exhaustion marker on these cells. Blocking PD-1 signaling in vitro restored the CD8+ T-cell functional defect. In addition, we showed that chronic SCI mice had higher levels of splenic NE, which contributed to the T-cell exhaustion phenotype, as PD-1 expression on both CD4+ and CD8+ T-cells was up-regulated following sustained exposure to NE in vitro.. These studies indicate that alteration of sympathetic activity following chronic SCI induces CD8+ T-cell exhaustion, which in turn impairs T-cell function and contributes to immune depression. Inhibition of the exhaustion pathway should be considered as a new therapeutic strategy for chronic SCI-induced immune depression.

Topics: Animals; Apoptosis; Calcium Ionophores; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Proliferation; Cytokines; Disease Models, Animal; Female; Ionomycin; Mice; Mice, Inbred C57BL; Norepinephrine; Programmed Cell Death 1 Receptor; Spinal Cord Injuries; Tetradecanoylphorbol Acetate; Tyrosine 3-Monooxygenase; Up-Regulation

The overall objective of this study is to non-invasively image and assess tumor targeting and retention of directly labeled T-lymphocytes following their adoptive transfer in mice. T-lymphocytes obtained from draining lymph nodes of 4T1 (murine breast cancer cell) sensitized BALB/C mice were activated in-vitro with Bryostatin/Ionomycin for 18 hours, and were grown in the presence of Interleukin-2 for 6 days. T-lymphocytes were then directly labeled with 1,1-dioctadecyltetramethyl indotricarbocyanine Iodide (DiR), a lipophilic near infrared fluorescent dye that labels the cell membrane. Assays for viability, proliferation, and function of labeled T-lymphocytes showed that they were unaffected by DiR labeling. The DiR labeled cells were injected via tail vein in mice bearing 4T1 tumors in the flank. In some cases labeled 4T1 specific T-lymphocytes were injected a week before 4T1 tumor cell implantation. Multi-spectral in vivo fluorescence imaging was done to subtract the autofluorescence and isolate the near infrared signal carried by the T-lymphocytes. In recipient mice with established 4T1 tumors, labeled 4T1 specific T-lymphocytes showed marked tumor retention, which peaked 6 days post infusion and persisted at the tumor site for up to 3 weeks. When 4T1 tumor cells were implanted 1-week post-infusion of labeled T-lymphocytes, T-lymphocytes responded to the immunologic challenge and accumulated at the site of 4T1 cell implantation within two hours and the signal persisted for 2 more weeks. Tumor accumulation of labeled 4T1 specific T-lymphocytes was absent in mice bearing Meth A sarcoma tumors. When lysate of 4T1 specific labeled T-lymphocytes was injected into 4T1 tumor bearing mice the near infrared signal was not detected at the tumor site. In conclusion, our validated results confirm that the near infrared signal detected at the tumor site represents the DiR labeled 4T1 specific viable T-lymphocytes and their response to immunologic challenge can be imaged in vivo.

Topics: Animals; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Cell Membrane; Cell Survival; Cell Tracking; Disease Models, Animal; Female; Immunohistochemistry; Immunotherapy, Adoptive; Interferon-gamma; Interleukin-2; Ionomycin; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Spectroscopy, Near-Infrared; T-Lymphocytes; Transplantation, Homologous

Does the application of three different artificial activating stimuli lead to a difference in pre- and post-implantation embryo development in the wobbler mouse, a mouse model with oocyte activation deficient round-headed sperm cells similar to human globozoospermia?. No gross differences were found between strontium chloride, electrical pulses or ionomycin with respect to the pre- and post-implantation development in the wobbler mouse.. Fertilization failure following intra-cytoplasmic sperm injection (ICSI) occurs in 1-3% of the ICSI cycles in human assisted reproduction technology (ART) and has been successfully overcome by different artificial activating stimuli. No comparison has been made yet in terms of their efficiency and safety.. Calcium release and embryo development were compared between oocytes fertilized by wobbler and wild-type (WT) sperm following ICSI with or without three different artificial activating agents. Preimplantation development was assessed on 70 injected oocytes on average per group. On average, 10 foster mothers were used per activating group to compare post-implantation development.. We used the wobbler mouse model that possesses oocyte activation deficient round-headed sperm cells. First, the calcium release following ICSI using wobbler sperm was compared with that of WT sperm. Outcome measures were the percentage of oocytes that showed calcium release and their mean amount of calcium rises. Secondly, the pre- and post-implantation development was assessed following ICSI with wobbler sperm plus artificial oocyte activation using either: (i) strontium chloride (Wob-Sr), (ii) electrical pulses (Wob-E) or (iii) ionomycin (Wob-I). Outcome measures were the activation, cleavage and blastocyst rates and the assessment of blastocyst quality by differential staining. Following mouse embryo transfer, pregnancy and birth rates as well as mean litter sizes were examined. Finally, pups were followed up until 8 weeks of age and then mated with fertile controls to assess their fertility.. The percentage of oocytes showing calcium rises as well as the number of calcium rises per oscillating oocyte were significantly lower in the wobbler group when compared with the WT group (9.3 versus 96% and 2.1 calcium rises versus 31 calcium rises) (P < 0.001). The fertilization rate was significantly lower in the wobbler group (11.4%) when compared with the WT group (92.1%) and the artificial activation groups (strontium chloride: 99%, electrical pulses: 99% and ionomycin: 81%, respectively) (P < 0.001). Post-implantation development did not differ significantly between the WT and artificial activation groups, with pregnancy rates in favor of strontium chloride and electrical pulses. The weight of the male pups did not differ between the study groups, whereas the weight of the female pups originating from Wob-Sr embryos was significantly lower at weeks 2, 3 and 4 when compared with female pups originating from WT embryos. However, the latter difference was not observed at later time points, nor in the other artificial activating groups. All offspring mated successfully with fertile controls.. Results in animal models should be extrapolated with caution to a subfertile human population. Also, ionomycin is currently the most widely used artificial oocyte activating agent in human ART.. The low frequency of observed calcium rises and the low activation rate make the wobbler mouse a highly suitable model to study oocyte activation deficiency. Strontium chloride and electrical pulses were more efficient means to restore fertilization rates and to support pre- and post-implantation embryonic development than ionomycin.. This work was supported by the Flemish foundation of Scientific Research (FWO-Vlaanderen) (aspirant clinical research mandate to F.V.M., fundamental clinical research mandate to P.D.S.); and Ghent University grant (KAN-BOF E/01321/01 to B.H.). The authors have no competing interests to declare.

Topics: Animals; Blastocyst; Calcium; Disease Models, Animal; Electrophysiology; Embryo Implantation; Embryo Transfer; Female; Infertility, Male; Ionomycin; Male; Mice; Oocytes; Pregnancy; Pregnancy, Animal; Sperm Injections, Intracytoplasmic; Spermatozoa; Strontium

Schistosomiasis japonica is a severe tropical disease caused by the parasitic worm Schistosoma japonicum. Among the most serious pathological effects of S. japonicum infection are hepatic lesions (cirrhosis and fibrosis) and portal hypertension. Interleukin-17 (IL-17) is a pro-inflammatory cytokine involved in the pathogenesis of many inflammatory and infectious conditions, including schistosomiasis. We infected C57BL/6 mice with S. japonicum and isolated lymphocytes from the liver to identify cell subsets with high IL-17 expression and release using flow cytometry and ELISA. Expression and release of IL-17 was significantly higher in hepatic lymphocytes from infected mice compared with control mice in response to both non-specific stimulation with anti-CD3 monoclonal antibody plus/anti-CD28 monoclonal antibody and PMA plus ionomycin. We then compared IL-17 expression in three hepatic T-cell subsets, T helper, natural killer T and γδT cells, to determine the major source of IL-17 during infection. Interleukin-17 was induced in all three subsets by PMA + ionomycin, but γδT lymphocytes exhibited the largest increase in expression. We then established a mouse model to further investigate the role of IL-17 in granulomatous and fibrosing inflammation against parasite eggs. Reducing IL-17 activity using anti-IL-17A antibodies decreased infiltration of inflammatory cells and collagen deposition in the livers of infected C57BL/6 mice. The serum levels of soluble egg antigen (IL)-specific IgGs were enhanced by anti-IL-17A monoclonal antibody blockade, suggesting that IL-17 normally serves to suppress this humoral response. These findings suggest that γδT cells are the most IL-17-producing cells and that IL-17 contributes to granulomatous inflammatory and fibrosing reactions in S. japonicum-infected C57BL/6 mouse liver.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neutralizing; Antibodies, Protozoan; Cells, Cultured; Collagen Type III; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Granuloma; Inflammation Mediators; Interleukin-17; Ionomycin; Liver; Liver Cirrhosis; Mice; Mice, Inbred C57BL; Natural Killer T-Cells; Receptors, Antigen, T-Cell, gamma-delta; Schistosoma japonicum; Schistosomiasis japonica; T-Lymphocyte Subsets; Th17 Cells; Up-Regulation

Animal-models are the basis of DC-based human immunotherapies. We describe the standardization of a canine-DC-generation protocol using different cytokines and characterize the quality and functional repertoire of the obtained canine-DCs. DCs were generated from healthy dog-PBMCs under serum-free and serum-containing conditions. DC-quality and -quantity was determined by FACS studying the expression-profiles of DC-/costimulatory- and maturation-antigens before/after culture with canine and human monoclonal-antibodies (cmabs/hmabs). Individual DCAgs-(DC-antigens)-expression-profiles were found before and after culture depending on the agents' mode-of-action. With at least one of three serum-free methods (Ca-Ionophore, Picibanil, Cytokines) sufficient DC-amounts were generated. So, canine-DCs can be regularly generated under serum-free conditions and hmabs additionally to cmabs qualify for staining/quantification of canine-cells/DCs. The canine-DCs were functional, shown by T-cell-activation, -proliferation and antigen-specific CTL-responses. In summary, successful, quantitative DC-generation is possible with serum-free methods. DC-based T-cell-vaccination-strategies evaluated for e.g. AML-patients can be tested in the dog and estimated in clinical studies for DC-vaccination-strategies.

Topics: Animals; Cell Culture Techniques; Cell Separation; Cells, Cultured; Culture Media, Serum-Free; Cytokines; Dendritic Cells; Disease Models, Animal; Dogs; Female; Flow Cytometry; Immunotherapy; Ionomycin; Male; Picibanil

Oxidized phospholipids (Ox-PLs) are generated in abundance at sites of inflammation. Recent studies have indicated that Ox-PLs may also exhibit anti-inflammatory activities. In this study, we investigated the beneficial effect of VB-201, a pure synthetic Ox-PL analog that we synthesized, on the development of a central nervous system (CNS) autoimmune inflammatory disease, in vivo. Oral administration of VB-201 ameliorated the severity of experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) peptide MOG35-55, and restrained the encephalogenicity of MOG35-55-specific T-cells. Our data presents a novel prospect for the role of Ox-PL analogs in CNS inflammatory diseases.

Topics: Animals; Bone Marrow Cells; Bromodeoxyuridine; CD4 Antigens; Cell Differentiation; Central Nervous System; Cytokines; Dendritic Cells; Disease Models, Animal; Drug Interactions; Encephalitis; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Female; Forkhead Transcription Factors; Freund's Adjuvant; Glycerylphosphorylcholine; Glycoproteins; Ionomycin; Ionophores; Lymph Nodes; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Pertussis Toxin; Phosphorylcholine; Polymethacrylic Acids; Severity of Illness Index; T-Lymphocytes; Time Factors

Neural-cadherin (N-cadherin), a member of the classical cadherin family of transmembrane glycoproteins, mediates cellular recognition and cell-cell adhesion through calcium-dependent homophilic interactions and plays important roles in the development and maintenance of the nervous system. Metalloproteinase is known to cleave N-cadherin, which is further cleaved by gamma-secretase. The intracellular domain of N-cadherin interacts with beta-catenin, and beta-catenin stability is critical for cell-cell adhesion and cell survival. In the present study, we showed that N-cadherin is cleaved specifically by calpain, resulting in the generation of a novel 110 kDa fragment. The cleavage occurred in ischemic brain lesions and in vitro neural cells in the presence of NMDA and ionomycin, and was restored by calpain inhibitors but not matrix metalloproteinase or gamma-secretase inhibitors. Calpain directly cleaved N-cadherin in in vitro calpain assays, and calpain inhibitors prevented its cleavage in a dose-dependent manner. Using N-cadherin deletion mutants, we found that calpain cleavage sites exist in at least four regions of the cytoplasmic domain. Treatment with NMDA induced neuronal death, and it suppressed the expression of surface N-cadherin and the N-cadherin/beta-catenin interaction, effects that were prevented by calpain inhibitor. Furthermore, calpain-mediated N-cadherin cleavage significantly affected cell-cell adhesion, AKT signaling, the N-cadherin/beta-catenin interaction and the Wnt target gene expressions through the accumulation of nuclear beta-catenin.

Topics: Animals; Animals, Newborn; beta Catenin; Biotinylation; Brain Injuries; Cadherins; Calcium; Calpain; Cells, Cultured; Disease Models, Animal; Dizocilpine Maleate; Embryo, Mammalian; Endocytosis; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Gene Expression Regulation; Green Fluorescent Proteins; In Vitro Techniques; Ionomycin; Ionophores; Mice; Models, Biological; Mutation; N-Methylaspartate; Neurons; Protein Structure, Tertiary; Proto-Oncogene Proteins c-akt; Signal Transduction; Subcellular Fractions; Transfection; Wnt Proteins

Intermittent hypoxia (IH) resulting from sleep apnea can lead to pulmonary hypertension (PH) and right heart failure, similar to chronic sustained hypoxia (CH). Supplemental CO(2), however, attenuates hypoxic PH. We therefore hypothesized that, similar to CH, IH elicits PH and associated increases in arterial endothelial nitric oxide synthase (eNOS) expression, ionomycin-dependent vasodilation, and receptor-mediated pulmonary vasoconstriction. We further hypothesized that supplemental CO(2) inhibits these responses to IH. To test these hypotheses, we measured eNOS expression by Western blot in intrapulmonary arteries from CH (2 wk, 0.5 atm), hypocapnic IH (H-IH) (3 min cycles of 5% O(2)/air flush, 7 h/day, 2 wk), and eucapnic IH (E-IH) (3 min cycles of 5% O(2), 5% CO(2)/air flush, 7 h/day, 2 wk) rats and their respective controls. Furthermore, vasodilatory responses to the calcium ionophore ionomycin and vasoconstrictor responses to the thromboxane mimetic U-46619 were measured in isolated saline-perfused lungs from each group. Hematocrit, arterial wall thickness, and right ventricle-to-total ventricle weight ratios were additionally assessed as indexes of polycythemia, arterial remodeling, and PH, respectively. Consistent with our hypotheses, E-IH resulted in attenuated polycythemia, arterial remodeling, RV hypertrophy, and eNOS upregulation compared with H-IH. However, in contrast to CH, neither H-IH nor E-IH increased ionomycin-dependent vasodilation. Furthermore, H-IH and E-IH similarly augmented U-46619-induced pulmonary vasoconstriction but to a lesser degree than CH. We conclude that maintenance of eucapnia decreases IH-induced PH and upregulation of arterial eNOS. In contrast, increases in pulmonary vasoconstrictor reactivity following H-IH are unaltered by exposure to supplemental CO(2).

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Carbon Dioxide; Chronic Disease; Disease Models, Animal; Dose-Response Relationship, Drug; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypocapnia; Hypoxia; Ionomycin; Ionophores; Male; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oxygen; Polycythemia; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

Visceral leishmaniasis (VL) produced in BALB/c mice through intracardial administration of Leishmania donovani amastigotes was accompanied by hepatosplenomegaly with high organ parasite load and lymphadenopathy when followed up to 4-months or so. To elucidate the mechanism of immunosuppression associated with VL, we report here progressive impairment of the proliferative response of lymph node cells (lymphocytes) from infected animals (I-LNC) to in vitro stimulation with the combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin (Io) that could be related to the downregulation of PKC and MAP kinase (ERK 1/2) activation process. Further, pretreatment of I-LNC with the protein phosphatase inhibitor okadaic acid (OA), but not with calyculin A or sodium orthovanadate, significantly restored their proliferative response as well as PMA-induced activation of PKC. A population of LNC (primarily T-lymphocytes) from chronically infected animals was shown to undergo apoptosis, the number of which increased considerably following PMA+ Io stimulation. The apoptotic pathway, which was followed through binding of cells to Annexin V, activation of caspase-3 and fragmentation of DNA, involved destabilization of mitochondria, probably as a result of downregulation of PKC and Bcl-2. Interestingly, prior incubation of I-LNC with OA reversed the state of cell cycle arrest (anergy) and apoptosis through progression of cells from G0/G1 to S and G2/M phases with transcriptional activation of IL-2 and IL-2R genes. Our results suggest that the cellular (immune) dysfunction in VL could be attributed to dephosphorylation of key molecules in the T-lymphocyte signaling pathway by Ser/Thr phosphatase leading to their inactivation.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Cell Proliferation; Chronic Disease; Clonal Anergy; Cricetinae; Disease Models, Animal; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Ionomycin; Ionophores; Leishmania donovani; Leishmaniasis, Visceral; Lymph Nodes; Lymphocyte Activation; Lymphocytes; Mice; Mice, Inbred BALB C; Okadaic Acid; Phosphoprotein Phosphatases; Protein Kinase C; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tetradecanoylphorbol Acetate

Calpains are calcium-dependent intracellular nonlysosomal proteases that are believed to hydrolyze specific substrates important in calcium-regulated signaling pathways. Recently, an atypical member of the calpain family, calpain 10, was described, and genetic variation in this gene was associated with an increased risk of type II diabetes mellitus in humans. In the present report, a polyclonal antibody directed against rat calpain 10 was developed. This antibody was used to monitor the expression of calpain 10 protein in tissues from rats, mice, and humans. Calpain 10 protein was found to be present in all tissues examined by Western blotting including the lens, retina, brain, heart, and skeletal muscle. Although some calpain 10 was detectable in the water-soluble protein fraction of these tissues, it was preferentially found in the water-insoluble fraction. In the lens, immunohistochemistry revealed that calpain 10 was predominately located in the cytoplasm of epithelial and newly differentiating lens fibers at the transition zone. However, calpain 10 was found to be associated with the plasma membrane of differentiated lens fiber cells and the sarcolemma of skeletal muscle. In the lens epithelium-derived cell line, alphaTN4-1, the calpain 10 protein was found in a punctate distribution in the cell nucleus as well as the cytoplasm. After the elevation of intracellular calcium levels with ionomycin, calpain 10 protein levels in the nucleus of alphaTN4-1 cells increased markedly, whereas those in the cytoplasm decreased. In the lens, the elevation of intracellular calcium levels after selenite administration resulted in increased levels of calpain 10 RNA within 1 day and a loss of calpain 10 protein from the lens nucleus coincident with the onset of selenite cataract. In conclusion, calpain 10 seems to be a ubiquitous calpain, the expression level and subcellular distribution of which are dynamically influenced by calcium.

Topics: Adult; Age Factors; Aged; Aged, 80 and over; Animals; Baculoviridae; Blotting, Western; Brain; Calcium; Calpain; Cataract; Cell Line; Cell Nucleus; Child; Child, Preschool; Cloning, Molecular; Cytoplasm; Disease Models, Animal; DNA, Complementary; Humans; Immunoblotting; Immunohistochemistry; Ionomycin; Ionophores; Lens, Crystalline; Mice; Microscopy, Fluorescence; Middle Aged; Muscle, Skeletal; Myocardium; Protein Structure, Tertiary; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Retina; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Sarcolemma; Time Factors; Tissue Distribution

Topics: Animals; Calcium; Calpain; Cysteine Proteinase Inhibitors; Disease Models, Animal; Hypoxia; In Vitro Techniques; Ionomycin; Ionophores; Kidney Tubules, Proximal; L-Lactate Dehydrogenase; Proteins; Rats

Mesangial cells (MC) are considered to play an important role in the development of hypertension. The purpose of this study was to characterize the effects of cytosolic Ca2+ on membrane voltage and conductance of MC using stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar Kyoto rats (WKY). We applied the patch-clamp technique in the whole-cell configuration to measure membrane potential (Vm) and ion currents. There was no significant difference in resting Vm values between MC from WKY and SHRSP. The cytosolic Ca2+ increase induced membrane depolarization and the increase of Cl- currents in MC from WKY but not in MC from SHRSP. On the other hand, the Ca2+ increase induced membrane hyperpolarization and the increase of K+ currents in MC from SHRSP but not in MC from WKY. Such differences between MC from two rat strains may play an important role in the alterations in renal hemodynamics observed in hypertension.

Topics: Animals; Calcium; Cells, Cultured; Cerebrovascular Disorders; Chloride Channels; Disease Models, Animal; Electric Conductivity; Glomerular Mesangium; Hypertension; Ionomycin; Membrane Potentials; Patch-Clamp Techniques; Potassium Channels; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Time Factors

Severe combined immunodeficient (scid) mice engrafted with small pieces of full thickness gut wall from immunocompetent syngenic donors develop a chronic and lethal colitis. Lymphocytes from the lamina propria of engrafted mice were analyzed for phorbol ester/ionomycin-induced cytokine production by intracellular staining. A 4-5-fold increase in the fraction of IFN-gamma-producing CD4+ lamina propria T cells was found in moderately and severely diseased mice when compared to healthy congenic C.B-17 control mice. The number of IL-2-producing T cells was increased by approximately 2-fold when comparing mice suffering from severe disease to healthy control mice. The fraction of TNF-alpha positive CD4+ T cells was increased by a factor of two in both moderately and severely diseased mice. When analyzing Th2 cytokines, it was found that the levels of IL-4-producing CD4+ T cells was not altered in diseased animals, whereas the fraction IL-10-producing CD4+ T cells was reduced by a factor of 20. The combined data showed a 15-25-fold increase in the Th1/Th2 ratio of diseased mice when compared to healthy control mice. No intracellular cytokines could be detected in lymphocytes not treated with phorbol ester/ionomycin. The present data identify a prominent role for Th1-type T helper cells in the immunopathogenesis of gut wall graft-induced inflammatory bowel disease in scid mice.

Topics: Animals; Colon; Disease Models, Animal; Inflammatory Bowel Diseases; Interferon-gamma; Interleukin-10; Interleukin-4; Intestines; Ionomycin; Lymphokines; Mice; Mice, SCID; Tetradecanoylphorbol Acetate; Th1 Cells; Th2 Cells; Transplantation, Heterotopic; Tumor Necrosis Factor-alpha

Experimental traumatic brain injury (TBI) damages cerebral vascular endothelium and reduces cerebral blood flow (CBF). The nitric oxide synthase (NOS) substrate, L-arginine, prevents CBF reductions after TBI, but the mechanism is not known. This study examined the possibility that post-traumatic hypoperfusion is due to reductions in the substrate sensitivity of NOS which are overcome by L-arginine. Isoflurane-anesthetized rats were prepared for TBI (midline fluid-percussion, 2.2 atm), sham-TBI, or no surgery (control), and were decapitated 30 min after injury or sham injury. The brains were removed and homogenized or minced for measurements of crude soluble or cell-dependent stimulated NOS activity, respectively. Baseline arterial oxygen, carbon dioxide, pH, or hemoglobin levels did not differ among control, sham, or TBI groups. Total cortical soluble NOS activity in TBI-treated rats was not significantly different from either untreated or sham groups when 0.45 microM or 1.5 microM L-arginine was added. Also, there were no differences in cell-dependent NOS activity among the three groups stimulated by 300 microM N-methyl-D-aspartate, 50 mM K+, or 10 microM ionomycin. These data suggest that TBI reduces CBF by a mechanism other than altering the substrate specificity or activation of nNOS.

Topics: Analysis of Variance; Animals; Arginine; Blood Pressure; Brain Injuries; Cerebral Cortex; Cerebrovascular Circulation; Disease Models, Animal; Excitatory Amino Acid Agonists; Ionomycin; Ionophores; Male; N-Methylaspartate; Nitric Oxide Synthase; Potassium Chloride; Rats; Rats, Sprague-Dawley

The authors have previously reported that homologous immunoglobulin (Ig)G reduces the occurrence of dextran sulfate sodium (DSS)-induced colitis, mainly by suppressing recruitment of immunocompetent cells into colitis lesions. However, the mechanisms of cell recruitment and of its suppression by IgG remain unclear. In addressing these questions, this study focused on the activation of T cells in the presence of macrophages. The authors found that [3H]-thymidine uptake of T cells from DSS-induced colitis mice, but not from normal mice, was significantly enhanced when cultured with DSS-pulsed macrophages. From the profile of cytokine production, it was suggested that T helper 1 (Th1)-type cells become predominant during stimulation. Addition of homologous IgG significantly suppressed T cell proliferation in a dose-dependent manner, while no suppressive effect was observed with heterologous IgG. Mouse IgG F(ab')2, but not Fc, fragments partially mimicked the suppressive effect of whole IgG. These findings provide evidence that Th1-type cells may play an important role in the development of DSS-induced colitis and that homologous IgG exerts its protective action at least in part through the F(ab')2 portion.

Topics: Animals; Antibodies, Monoclonal; CD3 Complex; Cell Division; Colitis; Cytokines; Dextran Sulfate; Disease Models, Animal; Female; Humans; Immunoglobulin G; Ionomycin; Isoantigens; Lymphocyte Activation; Macrophages; Mice; Mice, Inbred BALB C; Mice, Inbred CBA; Mitogens; T-Lymphocytes; Tetradecanoylphorbol Acetate

1. The aim of the present study was to test whether the vitamin D-dependent Ca(2+)-binding protein calbindin-D9k could function as an important cytosolic Ca2+ buffer in duodenal enterocytes while facilitating transepithelial active transport of Ca2+ ions. For the investigations we used dual-wavelength, fluorescence ratio imaging, with fura-2 as the Ca(2+)-sensitive dye, to measure changes in cytosolic concentrations of free Ca2+ ions ([Ca2+]i) in isolated pig duodenal enterocytes affected by different cytosolic calbindin-D9k concentrations. 2. Epithelial cells were obtained from weaned piglets with normal calbindin-D9k concentrations (con-piglets), from piglets with low calbindin-D9k levels due to inherited calcitriol deficiency caused by defective renal 25-hydroxycholecalciferol D3-1 alpha-hydroxylase activity (def-piglets), and from piglets with reconstituted calbindin-D9k concentrations, i.e. def-animals treated with high doses of vitamin D3 which elevated plasma calcitriol levels by extrarenal production (def-D3-piglets). Basal levels of [Ca2+]i ranged between 170 and 205 nM and did not differ significantly between the groups. 3. After addition of 5 mM theophylline, the [Ca2+]i in enterocytes from con-piglets doubled during the 10 min incubation. This effect, however, was three times higher in enterocytes from def-piglets compared with those from con-piglets. Similar results were obtained after 4 min incubation of enterocytes from con- and def-piglets in the presence of 1 microM ionomycin. In preparations from def-D3-piglets, ionomycin-induced increases in [Ca2+]i were significantly lower compared with enterocytes from def-piglets and were not different from the control values. 4. From the results, substantial support is given for the hypothesis that one of the major functions of mucosal calbindin-D9k is the effective buffering of Ca2+ ions.

Topics: Animals; Biological Transport, Active; Calbindins; Calcitriol; Calcium; Cell Survival; Cholecalciferol; Cytosol; Disease Models, Animal; Duodenum; Female; Intestinal Mucosa; Ionomycin; Ionophores; Male; Microvilli; S100 Calcium Binding Protein G; Swine; Theophylline; Vitamin D Deficiency

Systemic lupus erythematosus is characterized by polyclonal B cell activation, the production of autoantibodies, and often by renal disease. Previous studies demonstrated that unfractionated B cells from several strains of mice with lupus hyperproliferate in culture when stimulated with lipopolysaccharide (LPS) or anti-IgM. We wished to further examine proliferation of resting B cells from the BXSB mouse model of lupus and mice with the Yaa allele, when activated with a number of stimuli. Our work demonstrates that: (1) resting B cells from mice containing the Yaa allele hyperproliferated compared to that seen with B cells from mice lacking the Yaa allele, (2) this hyperproliferation occurred whether cells were stimulated with phorbol myristate acetate/ionomycin, LPS, anti-IgM, or CD40L cross-linking, (3) this hyperproliferation is specific to B and not T cells. Taken together these data suggest that one mechanism by which the Yaa allele contributes to the accelerated onset of lupus in BXSB male mice is through its influence on B cell activation.

Topics: Alleles; Animals; Antibodies, Anti-Idiotypic; B-Lymphocytes; Disease Models, Animal; Genetic Linkage; Immunoglobulin M; Ionomycin; Lipopolysaccharides; Lupus Erythematosus, Systemic; Lymphocyte Activation; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Spleen; Tetradecanoylphorbol Acetate; Time Factors

The cellular immune response was evaluated in a C3H/HeJ mouse model of intestinal amebiasis at 5-60 days postinoculation with Entamoeba histolytica. At various intervals, spleen lymphocytes were obtained from infected and noninfected control mice and cultured with concanavalin A (Con A), pokeweed mitogen (PWM), or ameba antigen. E. histolytica infection induced a cyclic depression of DNA synthesis when spleen lymphocytes were stimulated with a T-cell mitogen (Con A), a T- and B-cell (PWM) mitogen, or an ameba antigen. A similar response was observed in the determinations of interleukin-2 in the supernatants of Con A-stimulated spleen cells from infected mice. When spleen cells from E. histolytica-infected mice were stimulated with phorbol myristate acetate plus ionomycin, results indicated a signal-transduction defect. These alterations, observed at the cellular level, might facilitate invasion of the host by the parasite.

Topics: Animals; Antigens, Protozoan; Disease Models, Animal; Entamoeba histolytica; Entamoebiasis; Immunity, Cellular; Interleukin-2; Intestinal Diseases, Parasitic; Ionomycin; Lymphocyte Activation; Mice; Mice, Inbred C3H; Mitogens; Signal Transduction; Spleen; Tetradecanoylphorbol Acetate