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Vitro Proliferation (vitro + proliferation)
Selected AbstractsEnamel matrix derivative exhibits angiogenic effect in vitro and in a murine modelJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 8 2003Kuo Yuan Abstract Objectives: Angiogenesis is one of the most critical events in the wound healing process. Any increase in angiogenesis could result in more rapid and complete healing. A recent study found that enamel matrix derivative (EMD) could accelerate early periodontal wound healing. We wanted to clarify whether EMD caused an angiogenic effect and, thus, possibly enhanced wound healing. Methods: We performed in vitro proliferation and chemotaxis assays on human umbilical vein endothelial cell (HUVEC) cultures, and a tissue culture assay using blood vessel fragments in fibrin gel. Collagen membranes soaked with EMD were implanted subcutaneously in mice to test the in vivo angiogenic effect. Results: While there were no significant differences between the negative control and EMD groups in the proliferation assay, EMD treatment did exhibit a significantly greater dose-dependent chemotactic effect on HUVEC than control group treatments. The tissue culture in fibrin gel showed new blood vessel outgrowths in the EMD groups, but none in the negative control group. In the animal studies, significantly more endothelial cells were detected in the EMD group of mice. Conclusions: Our findings show that EMD does exhibit some angiogenic effects. However, the underlying molecules and mechanisms are still unidentified. We discuss several possibilities. Zusammenfassung Ziele: Die Angiogenese gehört zu den kritischsten Ereignissen bei der Wundheilung. Eine Erhöhung der Angiogenese könnte zu einer rascheren und kompletteren Wundheilung führen. Kürzlich zeigte eine Studie, dass Schmelzmatrixderivate (EMD) die frühe parodontale Wundheilung beschleunigen könnte. Wir wollten klären, ob EMD einen angiogenetischen Effekt verursacht und dies möglicherweise die Wundheilung verbessert. Methoden: Wir führten in vitro Proliferations- und Chemotaxis-Assays an menschlichen Umbilicalvenen-Endothelzellen (HUVEC)Kulturen durch und studierten eine Gewebekultur unter Nutzung von Blutgefäßfragmenten in Fibringel. Kollagenmembranen mit EMD getränkt wurden subkutan in Mäuse implantiert, um den angiogenetischen Effekt in vivo zu testen. Ergebnisse: Während es keine signifikanten Differenzen zwischen den negativen Kontrollen und den EMD Gruppen in dem Proliferationsassay gab, zeigte die EMD Behandlung einen signifikant größeren, dosisabhängigen chemotaktischen Effekt auf HUVEC verglichen mit den Kontrollen. Die Gewebekultur im Fibringel zeigte neue Blutgefäßbildungen in den EMD-Gruppen, aber keine bei den Negativkontrollen. Bei den Tierstudien wurden signifikant mehr Endothelzellen in den EMD Mäusegruppen entdeckt. Schlussfolgerungen: Unsere Ergebnisse zeigen, dass EMD einige angiogenetische Effekte zeigt. Jedoch sind die zugrunde liegenden Moleküle und die Mechanismen noch nicht geklärt. Wir diskutieren verschiedene Möglichkeiten. Résumé Objectifs: L'Angiogenèse est un des plus critiques éléments lors du processus de cicatrisation. La moindre augmentation de l'angiogenèse peut entraîner une cicatrisation plus rapide et plus complète. Une récente étude a montré que les dérivés de la matrice amellaire (EMD) pouvait accélérer plus tôt la cicatrisation parodontale. Nous voulions clarifier la possible responsabilité de l'EMD dans l'angiogenèse et si oui, l'amélioration de la cicatrisation. Méthodes: Nous avons réalisé in vitro la prolifération et un essai de chimiotactisme sur des cultures de cellules endothéliales de la veine ombilicale humaine (HUVEC), et un essai de culture tissulaire en utilisant des fragments de vaisseaux sanguins dans un gel de fibrine. Des membranes de collagène gorgées d'EMD furent implantées en sous-cutanée chez des souris pour tester l'effet angiogénique in vivo. Résultats: Bien qu'il n'y eut pas de différences significatives entre le contrôle négatif et le groupe EMD pour le test de prolifération, le traitement par EMD présentait un effet chimiotactique dose- dépendant significativement plus élevé sur les HUVEC. La culture tissulaire sur gel de fibrine présentait une surcroissance de nouveaux vaisseaux sanguins pour le groupe EMD, mais pas dans le groupe contrôle. Plus de cellules endothéliales furent en outre détectées lors de l'étude animale, pour le groupe de souris traitées par EMD. Conclusions: Nos données montrent que l'EMD présente quelques effets angiogéniques. Cependant, les molécules et les mécanismes responsables ne sont toujours pas identifiés. Nous discutons quelques possibilités. [source] Inorganic arsenic induces necrosis of human CD34-positive haematopoietic stem cellsENVIRONMENTAL TOXICOLOGY, Issue 2 2008Laurent Vernhet Abstract Inorganic arsenic is a major environmental contaminant known to exert immunosuppressive effects. In this study, we report toxicity of As2O3, a trivalent inorganic form, toward isolated human hematopoietic CD34+ progenitor cells. Our results demonstrate that low concentrations of As2O3 (0.1,5 ,M) inhibit in vitro proliferation of CD34+ cells and their differentiation into various hematological cell lineages. These effects were associated with the induction of a necrotic process independent of caspases and likely related to mitochondrial damage. We conclude that As2O3 can impair in vitro human hematopoiesis by decreasing survival of CD34+ progenitor cells. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2008. [source] Effects of angiogenic regulators on in vitro proliferation and cytokine secretion by native human acute myelogenous leukemia blastsEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 1 2003Øystein Bruserud Abstract: Angiogenesis seems to be important in the pathogenesis of acute myelogenous leukemia (AML). The endothelial cell proliferation and microvessel formation are regulated by a wide range of soluble mediators, including angiogenin, angiopoietin-2, basic fibroblast growth factors, vascular endothelial growth factor (VEGF), VEGF-D, angiostatin and endostatin. In the present study, it has been investigated whether these mediators have an additional direct effect on the proliferation and cytokine release by native human AML blasts. AML cells derived from a large group of consecutive patients were investigated. All these mediators could alter the proliferation and cytokine release [interleukin (IL) 1,, IL6, IL8, tumor necrosis factor ,] for a minority of patients. Alteration of spontaneous proliferation by at least one mediator was detected in five of 38 patients; whereas, altered cytokine (Flt3-ligand, granulocyte-macrophage colony-stimulating factor, stem cell factor)-dependant proliferation was observed for 10 patients. Growth enhancement was most frequently observed, whereas growth inhibition was uncommon. The effects on AML blast proliferation were often dependant on or were modulated by the presence of the three hematopoietic growth factors. Based on the present results, it is concluded that angioregulatory mediators have additional growth-enhancing effects directly on the AML blasts for certain patients. However, based on the results from this investigation and previous studies it is suggested that their major contribution to the pathogenesis of AML is through their effects on regulation of bone marrow angiogenesis, and future studies of these mediators in AML should probably focus on these effects. [source] Suppressive properties of human CD4+CD25+ regulatory T,cells are dependent on CTLA-4 expressionEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 12 2004Brigitte Birebent Abstract It has been demonstrated that T,cells with regulatory properties are present within the peripheral blood CD4+CD25+ T,cell compartment. Here, we describe an original method to purify human CD4+CD25+CD152+ T,lymphocytes as living cells by forcing the exportation of CTLA-4 molecules stored in intracellular vesicules at the cell surface. By doing so, we demonstrate that CD4+CD25+ T,cells contain a smaller and more homogeneous population enriched in cells with in vitro regulatory activity. Moreover, we show that this enrichment in regulatory T,cells is associated with an increased expression of Foxp3 and that CD4+CD25+CD152+ T,lymphocytes display a much stronger suppressive activity in controlling in vitro proliferation of alloantigen-specific T,cells than CD4+CD25+CD152, T,lymphocytes purified in parallel. Lastly, by purifying such cells expressing CTLA-4, we demonstrate that indeed CTLA-4 is involved in CD4+CD25+CD152+ T,cell regulatory activity, while suppressive cytokines are not. [source] Functional characterization of T cells differentiated in vitro from bone marrow-derived CD34+ cells of psoriatic patients with family historyEXPERIMENTAL DERMATOLOGY, Issue 8 2010Kaiming Zhang Please cite this paper as: Functional characterization of T cells differentiated in vitro from bone marrow-derived CD34+ cells of psoriatic patients with family history. Experimental Dermatology 2010; 19: e128,e135. Abstract Background:, The strong but complex genetic background suggests that inherent and intrinsic rather than exogenous factors have a key role in immunopathogenesis of psoriasis. It is reasonable to speculate that the dysfunctional activity of psoriatic T cells may partly originate from the abnormal haematopoietic cells. Objectives:, To test if T cells originated from haematopoietic progenitor cells in psoriasis patients display functional alternations similar to previously reported abnormalities of circulating T cells. Methods:, Bone marrow CD34+ haematopoietic cells were isolated from psoriatic patients with family history and healthy subjects, and differentiated into T cells in vitro in the thymic stromal co-culture system. These cells were further subjected to functional comparisons such as in vitro proliferation, secretion of cytokines such as IL-4, IL-8 and IFN,,, and inducing the production of C-myc, Bcl-xL, and Ki67 proteins in human keratinocytes. Results:, While bone marrow-derived CD34+ cells from both patients and healthy volunteers developed into mature T cells within weeks in the thymic environment in vitro, the differentiated T cells from psoriatic patients showed higher proliferation and stronger capacity to secret TH1 cytokines in response to streptococcal superantigen. The differentiated T cells from psoriatic patients, but not from normal controls, induced overexpression of C-myc and Ki67, but not Bcl-XL, in keratinocytes. Conclusions:, T cells differentiated from CD34+ cells of psoriatic patients, but not normal controls, are functionally similar to psoriatic circulating T cells, suggesting that the dysfunctional activity of T cells in psoriatic patients can be traced back to the early development of haematopoietic cells. [source] Mechanism of T cell tolerance induction by murine hepatic Kupffer cells,HEPATOLOGY, Issue 3 2008Qiang You The liver is known to favor the induction of immunological tolerance rather than immunity. Although Kupffer cells (KC) have been indicated to play a role in liver tolerance to allografts and soluble antigens, the mechanisms involved remain unclear. We hypothesized that KCs could promote immune tolerance by acting as incompetent antigen-presenting cells (APC), as well as actively suppressing T cell activation induced by other potent APCs. The expression of antigen presentation-related molecules by KCs was phenotyped by flow cytometry. The abilities of KCs to act as APCs and to suppress T cell activation induced by splenic dendritic cells (DC) were examined by in vitro proliferation assays using CD4+ OVA-TCR (ovalbumin T cell receptor) transgenic T cells. We found that, compared with DCs, KCs expressed significantly lower levels of major histocompatibility complex (MHC) II, B7-1, B7-2, and CD40. This result is consistent with our observation that KCs were not as potent as DCs in eliciting OVA-specific T cell proliferation. However, KCs isolated from polyinosinic:polycytidylic acid,treated mice expressed significantly higher levels of MHC II and costimulatory molecules than did naïve KCs and could stimulate stronger T cell responses. More importantly, we found that KCs could inhibit DC-induced OVA-specific T cell activation. Further investigation of the underlying mechanism revealed that prostaglandins produced by KCs played an important role. The results ruled out the possible involvement of interleukin-10, nitric oxide, 2,3-dioxygenase, and transforming growth factor , in KC-mediated T cell suppression. Conclusion: Our data indicate that KCs are a tolerogenic APC population within the liver. These findings suggest that KCs may play a critical role in regulating immune reactions within the liver and contributing to liver-mediated systemic immune tolerance. (HEPATOLOGY 2008.) [source] Human liver stem cell-derived microvesicles accelerate hepatic regeneration in hepatectomized ratsJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 6b 2010M. B. Herrera Abstract Several studies indicate that adult stem cells may improve the recovery from acute tissue injury. It has been suggested that they may contribute to tissue regeneration by the release of paracrine factors promoting proliferation of tissue resident cells. However, the factors involved remain unknown. In the present study we found that microvesicles (MVs) derived from human liver stem cells (HLSC) induced in vitro proliferation and apoptosis resistance of human and rat hepatocytes. These effects required internalization of MVs in the hepatocytes by an ,4 -integrin-dependent mechanism. However, MVs pre-treated with RNase, even if internalized, were unable to induce hepatocyte proliferation and apoptosis resistance, suggesting an RNA-dependent effect. Microarray analysis and quantitative RT-PCR demonstrated that MVs were shuttling a specific subset of cellular mRNA, such as mRNA associated in the control of transcription, translation, proliferation and apoptosis. When administered in vivo, MVs accelerated the morphological and functional recovery of liver in a model of 70% hepatectomy in rats. This effect was associated with increase in hepatocyte proliferation and was abolished by RNase pre-treatment of MVs. Using human AGO2, as a reporter gene present in MVs, we found the expression of human AGO2 mRNA and protein in the liver of hepatectomized rats treated with MVs. These data suggested a translation of the MV shuttled mRNA into hepatocytes of treated rats. In conclusion, these results suggest that MVs derived from HLSC may activate a proliferative program in remnant hepatocytes after hepatectomy by a horizontal transfer of specific mRNA subsets. [source] Human articular chondrocytes suppress in vitro proliferation of anti-CD3 activated peripheral blood mononuclear cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2006Chiara Bocelli-Tyndall Objective: To investigate whether mature human articular chondrocytes (AC) exhibit an antiproliferative effect on activated peripheral blood mononuclear cells (PBMC) and to compare this effect with other cells of mesenchymal origin. Methods: AC from healthy cadaveric cartilage were grown for different passages, in the absence (control) or presence of factors enhancing cell de-differentiation (transforming growth factor (TGF),1, fibroblast growth factor (FGF)-2, and platelet derived growth factor (PDGF)bb-TFP medium). Cell ability to suppress PBMC proliferation driven by anti-CD3 antibody was measured by tritiated thymidine uptake following incubation for 48 h at different PBMC:AC ratios and expressed as percent of residual proliferation (RP). AC antiproliferative effect was compared to that of control dermal fibroblasts (DF) and bone marrow stromal cells (BMSC). Results: AC exhibited a cell number-dependent antiproliferative effect. The strongest effect (up to 2% RP) was measured using the least expanded AC cultures. The use of TFP medium for AC expansion resulted in a significantly lower antiproliferative effect, in the range of that induced by BMSC (up to 18% RP). Also DF induced a marked antiproliferative effect (up to 11% RP). Conclusion: We report for the first time that human AC have a marked antiproliferative effect on anti-CD3 stimulated PBMC, which is reduced upon culture in medium-inducing extensive cell de-differentiation. These results reflect the immunosuppressive properties observed for other different mesenchymal cell types and raise the question of a potential common physiological role in local tissue protection. J. Cell. Physiol. 209: 732,734, 2006. © 2006 Wiley-Liss, Inc. [source] Enamel matrix derivative exhibits angiogenic effect in vitro and in a murine modelJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 8 2003Kuo Yuan Abstract Objectives: Angiogenesis is one of the most critical events in the wound healing process. Any increase in angiogenesis could result in more rapid and complete healing. A recent study found that enamel matrix derivative (EMD) could accelerate early periodontal wound healing. We wanted to clarify whether EMD caused an angiogenic effect and, thus, possibly enhanced wound healing. Methods: We performed in vitro proliferation and chemotaxis assays on human umbilical vein endothelial cell (HUVEC) cultures, and a tissue culture assay using blood vessel fragments in fibrin gel. Collagen membranes soaked with EMD were implanted subcutaneously in mice to test the in vivo angiogenic effect. Results: While there were no significant differences between the negative control and EMD groups in the proliferation assay, EMD treatment did exhibit a significantly greater dose-dependent chemotactic effect on HUVEC than control group treatments. The tissue culture in fibrin gel showed new blood vessel outgrowths in the EMD groups, but none in the negative control group. In the animal studies, significantly more endothelial cells were detected in the EMD group of mice. Conclusions: Our findings show that EMD does exhibit some angiogenic effects. However, the underlying molecules and mechanisms are still unidentified. We discuss several possibilities. Zusammenfassung Ziele: Die Angiogenese gehört zu den kritischsten Ereignissen bei der Wundheilung. Eine Erhöhung der Angiogenese könnte zu einer rascheren und kompletteren Wundheilung führen. Kürzlich zeigte eine Studie, dass Schmelzmatrixderivate (EMD) die frühe parodontale Wundheilung beschleunigen könnte. Wir wollten klären, ob EMD einen angiogenetischen Effekt verursacht und dies möglicherweise die Wundheilung verbessert. Methoden: Wir führten in vitro Proliferations- und Chemotaxis-Assays an menschlichen Umbilicalvenen-Endothelzellen (HUVEC)Kulturen durch und studierten eine Gewebekultur unter Nutzung von Blutgefäßfragmenten in Fibringel. Kollagenmembranen mit EMD getränkt wurden subkutan in Mäuse implantiert, um den angiogenetischen Effekt in vivo zu testen. Ergebnisse: Während es keine signifikanten Differenzen zwischen den negativen Kontrollen und den EMD Gruppen in dem Proliferationsassay gab, zeigte die EMD Behandlung einen signifikant größeren, dosisabhängigen chemotaktischen Effekt auf HUVEC verglichen mit den Kontrollen. Die Gewebekultur im Fibringel zeigte neue Blutgefäßbildungen in den EMD-Gruppen, aber keine bei den Negativkontrollen. Bei den Tierstudien wurden signifikant mehr Endothelzellen in den EMD Mäusegruppen entdeckt. Schlussfolgerungen: Unsere Ergebnisse zeigen, dass EMD einige angiogenetische Effekte zeigt. Jedoch sind die zugrunde liegenden Moleküle und die Mechanismen noch nicht geklärt. Wir diskutieren verschiedene Möglichkeiten. Résumé Objectifs: L'Angiogenèse est un des plus critiques éléments lors du processus de cicatrisation. La moindre augmentation de l'angiogenèse peut entraîner une cicatrisation plus rapide et plus complète. Une récente étude a montré que les dérivés de la matrice amellaire (EMD) pouvait accélérer plus tôt la cicatrisation parodontale. Nous voulions clarifier la possible responsabilité de l'EMD dans l'angiogenèse et si oui, l'amélioration de la cicatrisation. Méthodes: Nous avons réalisé in vitro la prolifération et un essai de chimiotactisme sur des cultures de cellules endothéliales de la veine ombilicale humaine (HUVEC), et un essai de culture tissulaire en utilisant des fragments de vaisseaux sanguins dans un gel de fibrine. Des membranes de collagène gorgées d'EMD furent implantées en sous-cutanée chez des souris pour tester l'effet angiogénique in vivo. Résultats: Bien qu'il n'y eut pas de différences significatives entre le contrôle négatif et le groupe EMD pour le test de prolifération, le traitement par EMD présentait un effet chimiotactique dose- dépendant significativement plus élevé sur les HUVEC. La culture tissulaire sur gel de fibrine présentait une surcroissance de nouveaux vaisseaux sanguins pour le groupe EMD, mais pas dans le groupe contrôle. Plus de cellules endothéliales furent en outre détectées lors de l'étude animale, pour le groupe de souris traitées par EMD. Conclusions: Nos données montrent que l'EMD présente quelques effets angiogéniques. Cependant, les molécules et les mécanismes responsables ne sont toujours pas identifiés. Nous discutons quelques possibilités. [source] In vitro proliferation of axotomized rat facial nucleus-derived activated microglia in an autocrine fashionJOURNAL OF NEUROSCIENCE RESEARCH, Issue 2 2006Kazuyuki Nakajima Abstract Transection of rat adult facial nerve leads to an increase in the number of activated microglia in the facial nucleus (FN), with a peak in proliferation 3 days after transection. To investigate the characteristics of these activated microglia, we isolated the cells with high purity from axotomized FN (axFN) 3 days after transection according to the previously reported procedure for explant culture. The isolated microglia exhibited immunocytochemical properties similar to those in vivo, and their numbers increased approximately five- to sevenfold over a period of 10 days without the addition of any mitogens, suggesting that self-reproduction was occurring. Actually, the microglia actively incorporated bromodeoxyuridine (BrdU) and strongly expressed an S-phase-specific protein marker, proliferating cell nuclear antigen (PCNA). To examine the mechanism underlying this proliferation, the expression of the mitogens and specific receptors of the microglia were analyzed in conditioned medium (CM) and cells. Macrophage-colony stimulating factor (M-CSF) and granulocyte macrophage-CSF (GM-CSF) were detected in the CM as well as in the cells. Their specific receptor proteins, c-Fms and GMCSFR,, were also detected in the cell homogenate. These proliferating microglia were not found to produce deleterious factors for neurons. In summary, the microglia isolated from the axFN were found to be proliferative in an autocrine fashion and to have some cellular properties in common with those observed in vivo. © 2006 Wiley-Liss, Inc. [source] Pressureless Sintering and Mechanical and Biological Properties of Fluor-hydroxyapatite Composites with ZirconiaJOURNAL OF THE AMERICAN CERAMIC SOCIETY, Issue 12 2003Hae-Won Kim Fluor-hydroxyapatite (FHA) fabricated by a reaction between fluorapatite (FA) and hydroxyapatite (HA) was mixed with ZrO2 to produce FHA,ZrO2 composites. When the relative amount of FA to HA increased, the decomposition of the composite was decreased gradually because of the formation of thermally stable FHA solid solutions. With such suppression of decomposition, the FHA,ZrO2 composites retained fully densified bodies. As a result, significant enhancements in mechanical properties, such as hardness, flexural strength, and fracture toughness, were achieved as the relative amount of FA to HA increased. The highest values in strength and toughness were 220 MPa and 2.5 MPa·m1/2, respectively, with FHA,40 vol% ZrO2 composites. In vitro proliferation of osteoblast-like cells (MG63) on the composites showed behavior similar to that observed on pure HA and FHA. Alkaline phosphatase (ALP) activity of the growing cells (HOS) on the composites was slightly down-regulated compared with that on pure HA and FHA at prolonged periods. [source] Differences in lymphocyte gene expression between tolerant and syngeneic liver grafted ratsLIVER TRANSPLANTATION, Issue 3 2004Masayuki Fujino Induction of tolerance to allogeneic donor grafts is a clinically desirable goal in bone marrow and solid organ transplantation. We have taken the advantage of DNA microarray technology to investigate gene expression mechanism in regulatory cells. In the present study, using a tacrolimus (FK506) induced tolerance of the fully mismatched liver allograft rat model, we demonstrated that, in contrast with peripheral blood lymphocytes (PBLs) from syngeneic recipients, PBLs taken from tolerant recipients 100 days after transplantation were able to suppress the in vitro proliferation of allogeneic PBLs and to prolong the survival of second syngeneic recipients. We also compared messenger RNA profiles in PBLs from tolerant recipients with those from syngeneic recipients using a DNA microarray with probe sets corresponding to more than 8000 rat genes. There were 96 up-regulated and 103 down-regulated genes in the tolerant recipients. In the up-regulated group, there were 76 known genes and 20 expressed sequence tags (ESTs). In the down-regulated groups, there were 87 known genes and 16 ESTs. Our data indicated that FK506 treatment induced tolerance and expansion of regulatory cells and the DNA microarray technology was useful for this application and provided many informative insights into the mechanism of lymphocyte regulation. (Liver Transpl 2004;10:379,391.) [source] In vitro measurement of post-natal changes in proliferating satellite cell frequency during rat muscle growthANIMAL SCIENCE JOURNAL, Issue 2 2010Takahiro SUZUKI ABSTRACT Satellite cells, resident myogenic stem cells found in postnatal skeletal muscle, are most abundant during early postnatal development and sharply decline in frequency thereafter to adult levels in mice and rats. Therefore, postnatal changes in satellite cell mitotic activities are important aspects for further understanding a muscle growth strategy. In large meat-production animals, however, the traditional in vivo proliferation assay may be less realistic because it requires intra-peritoneal (ip) injection of huge dosage of mutagenic nucleosides, 3H-labeled thymidine or bromodeoxyuridine (BrdU), at each age-time of sacrifice. We report in the present pilot study using rats that in vivo proliferation activity of satellite cells can be evaluated by an in vitro BrdU-incorporation assay in early cultures. Briefly, satellite cells were prepared from upper hind-limb and back muscles and maintained for 24 h with imposing by BrdU addition for the last 2 h, followed by the regular immunocytochemistry for determining BrdU-incorporated cell percentage. This in vitro assay demonstrated a rapid decrease in proliferating satellite cell frequency to the adult level during about 3-month period after birth, and yielded a high correlation to the measurements by the in vivo BrdU ip-injection method during the postnatal period examined from day-2 to month-11. The in vitro proliferation assay may be further adaptable for large domestic animals by the combination with a muscle biopsy technique that enables age-interval sampling from the same growing animals. [source] Localizing central nervous system immune surveillance: Meningeal antigen-presenting cells activate T cells during experimental autoimmune encephalomyelitisANNALS OF NEUROLOGY, Issue 4 2009Pia Kivisäkk MD Objective The onset of neurological signs in experimental autoimmune encephalomyelitis is tightly associated with infiltration and reactivation of T cells in the central nervous system. The anatomic localization of the initial T cell-antigen-presenting cell (APC) interactions leading to reactivation of T cells in the central nervous system is, however, still unclear. We hypothesized that activated CD4+ T cells gain direct access to the subarachnoid space and become reactivated on encounter with cognate antigen in this compartment. Methods C57Bl/6 mice were immunized with MOG35-55, and interactions between CD4+ T cells and major histocompatibility class II+ APCs in the subarachnoid space were investigated using flow cytometry, confocal microscopy of leptomeningeal whole-mount preparations, time-lapse microscopy of leptomeningeal explants, and in vitro proliferation assays. Results CD4+ T cells, polarized to produce Th1/Th17 cytokines, accumulated in the subarachnoid space early during the course of experimental autoimmune encephalomyelitis, before CD4+ T cells were detected in the spinal cord parenchyma. At this time point, leptomeningeal but not parenchymal CD4+ T cells incorporated bromodeoxyuridine, indicating local proliferation of CD4+ T cells in the subarachnoid space. Time-lapse microscopy indicated that these CD4+ T cells actively scanned the tissue and interacted with local major histocompatibility class II+ APCs, resulting in long-lasting interactions between CD4+ T cells and major histocompatibility class II+ APCs, suggestive of immunological synapses. Interpretation These results support the concept that immune surveillance of the central nervous system involves the subarachnoid space and indicate that the leptomeninges play an important role in experimental autoimmune encephalomyelitis initiation. Ann Neurol 2009;65:457,469 [source] Functional and proteomic analysis of serum and cerebrospinal fluid derived from patients with traumatic brain injury: a pilot studyANZ JOURNAL OF SURGERY, Issue 7-8 2010Dieter Cadosch Abstract Background:, An enhanced fracture healing response has been reported in patients with traumatic brain injury (TBI). This has been attributed to circulating humoral factors that are thought to be proteins produced and released by the injured brain. However, these factors remain unknown. The aim of this study was to identify osteogenic factors in serum and cerebrospinal fluid (CSF) from TBI patients. This was carried out using in vitro proliferation assays with the human foetal osteoblastic 1.19 cell line (hFOB) combined with a novel proteomic approach. Methods:, Serum was collected from brain-injured (n = 12) and non-brain-injured (n = 9) patients with a comorbid femur shaft fracture. Similarly, CSF was obtained from TBI (n = 7) and non-TBI (n = 9) patients. The osteoinductive potential of these samples was determined by measuring the in vitro proliferation rate of hFOB cells. Highly osteogenic serum and CSF samples of TBI patients were chosen for protein analysis and were compared to those of non-brain-injured patients. A new hFOB cell-based method was used to enrich the proteins in these samples, which had a functional affinity for these osteoprogenitor cells. These enriched protein fractions were mapped using two-dimensional gel electrophoresis and protein imaging methods displaying serum and CSF proteins of brain-injured and control subjects that had an affinity for human osteoprogenitor cells. Results:, Serum and CSF derived from brain-injured patients demonstrated a greater osteoinductive potential (P < 0.05) than their non-brain-injured counterparts. Clear-cut differences in the pattern of proteins in two-dimensional gels were detected between TBI and control patients. Fourteen proteins were exclusively present in the serum of TBI patients, while other proteins were either up- or downregulated in samples collected from TBI patients (P < 0.05). Conclusion:, Osteoinductive factors are present in the serum and CSF of brain-injured patients. These may include one or more of those proteins identified as having an affinity for osteoprogenitor cells that are either exclusively present or up- or downregulated in the serum and CSF of brain-injured patients. [source] 1,,25-Dihydroxyvitamin D3 and its analogues, EB1089 and CB1093, profoundly inhibit the in vitro proliferation of the human hepatoblastoma cell line HepG2ANZ JOURNAL OF SURGERY, Issue 7 2001J. Akhter Background: 1,,25-dihydroxyvitamin D3 (1,25[OH]2D3) has been shown to inhibit the proliferation of various cancer cells including colon, prostate, melanoma, osteosarcoma and breast cancer. Methods: The human hepatoma cell line (HepG2) was cultured with 1,25(OH)2D3 or one of two analogues EB1089 or CB1093 for various durations. Cellular proliferation was measured by uptake of [3H]thymidine, and cell numbers were determined by trypan blue exclusion counting. Results: 1,25(OH)2D3, EB1089 and CB1093 all inhibited proliferation of HepG2 by up to 90% after 5 days of treatment, compared to the untreated controls. Decreased proliferation was associated with an approximately 50% reduction in cell numbers at concentrations of up to 10,10 mol/L after 5 days of treatment with 1,25(OH)2D3. Cell proliferation rapidly recovered in cultures treated with lower concentrations of 1,25(OH)2D3 (10,10 and 10,11 mol/L) when 1,25(OH)2D3 was removed from the cultures by placing cells in serum containing medium without 1,25(OH)2D3. When HepG2 cells were treated with 10,8 mol/L 1,25(OH)2D3 for 5 weeks, there was still significant inhibition of proliferation, although at week 5 there was 66% inhibition compared to 93% at the end of week 1. Conclusions: 1,25(OH)2D3, EB1089 and CB1093 all significantly inhibit the proliferation of HepG2 hepatoblastoma cells, with EB1089 being the most potent at lower concentrations. Inhibition can be maintained for at least 4 weeks, but is reversed after removal of vitamin D3. [source] Simultaneous signalling through c-mpl, c-kit and CXCR4 enhances the proliferation and differentiation of human megakaryocyte progenitors: possible roles of the PI3-K, PKC and MAPK pathwaysBRITISH JOURNAL OF HAEMATOLOGY, Issue 1 2001Hitoshi Minamiguchi We assessed the effect of signalling through CXCR4 on the proliferation and differentiation of human megakaryocytic progenitor cells (CFU-Meg) in the presence or absence of stem cell factor (SCF) and/or thrombopoietin (TPO), using peripheral blood-derived CD34+IL-6R, cells as a target. TPO alone induced a significant number of CFU-Meg colonies. Although stromal cell-derived factor-1 (SDF-1) or SCF alone did not support CFU-Meg colony formation, these factors had a synergistic effect on CFU-Meg colony formation in the presence of TPO. The combination of SDF-1, SCF and TPO induced twice as many CFU-Meg colonies as TPO alone. To investigate the mechanism of this synergistic action, we examined the effects of various protein kinase inhibitors on CFU-Meg colony formation. LY294002 and GF109203X (inhibitors of PI3-K and PKC respectively) completely or partially inhibited this synergistic action. In contrast, a MEK inhibitor (PD98059) did not inhibit CFU-Meg colony formation. It significantly increased the higher ploidy classes (16N to 64N) of megakaryocytes supported by TPO, TPO + SCF, TPO + SDF-1, and TPO + SCF + SDF-1, whereas it abolished the effect of SDF-1 on the increase of higher ploidy classes of megakaryocytes supported by TPO. These results suggest that MAPK may negatively or positively regulate the nuclear maturation of megakaryocytes, known as endomitosis. In the presence of PD98059, proplatelet formation (PPF) was significantly augmented, suggesting that the MAPK pathway may also inhibit the initiation of PPF. In conclusion, simultaneous activation of three signals through c-mpl, c-kit and CXCR4 can induce the in vitro proliferation and differentiation of CFU-Meg, and SDF-1 is a potentiator of human megakaryocytopoiesis. [source] 2C4, a monoclonal antibody against HER2, disrupts the HER kinase signaling pathway and inhibits ovarian carcinoma cell growthCANCER, Issue 12 2005Noriyuki Takai M.D. Abstract BACKGROUND Human epidermal growth factor receptor 2 (HER2) is overexpressed in 25,30% of ovarian carcinoma cases and a correlation between increased HER2 expression and decreased survival has been demonstrated. HER2 is a ligand-less member of the HER family that functions as the preferred coreceptor for epidermal growth factor receptor (EGFR), HER3, and HER4. METHODS An approach was developed to target HER2's role as a coreceptor using a monoclonal antibody, 2C4, which sterically hinders HER2's recruitment into a functional HER complex. RESULTS HER2 was robustly expressed in all eight ovarian carcinoma cell lines; expression of EGFR and HER3 was variable. Even though four of the eight cell lines responded to EGF, 2C4 antibody moderately inhibited in vitro proliferation of only two cell lines (OVCA433 and SK-OV-3). Furthermore, ligand-stimulated p-MAPK expression was inhibited by 2C4 only in these two cell lines after exposure to EGF. Immunoprecipitation and eTag analysis revealed that OVCA433 expressed heterodimers of EGFR/HER2, and these heterodimers were disrupted after treatment with 2C4, whereas OVCA432 cells did not have these heterodimers. In murine xenograft experiments, the in vivo growth of OVCA433, but not of OVCA432, ovarian carcinoma cells was significantly inhibited by 2C4 treatment of the mice. CONCLUSION 2C4 is able to disrupt the HER signaling pathway and inhibit the in vitro and in vivo growth of ovarian carcinoma cell lines. The response appears limited to lines in which HER2 heterodimers were able to transduce proliferative signals. Our findings suggest a strong rationale to conduct clinical trials of 2C4 in a subset of patients with ovarian tumors. Cancer 2005. © 2005 American Cancer Society. [source] |