Cells In Vitro (cell + in_vitro)

Distribution by Scientific Domains


Selected Abstracts


Role of the Latent Transforming Growth Factor ,,Binding Protein 1 in Fibrillin-Containing Microfibrils in Bone Cells In Vitro and In Vivo

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2000
Sarah L. Dallas
Abstract Latent transforming growth factor ,,binding proteins (LTBPs) are extracellular matrix (ECM) proteins that bind latent transforming growth factor , (TGF-,) and influence its availability in bone and other connective tissues. LTBPs have homology with fibrillins and may have related functions as microfibrillar proteins. However, at present little is known about their structural arrangement in the ECM. By using antibodies against purified LTBP1, against a short peptide in LTBP1, and against epitope-tagged LTBP1 constructs, we have shown colocalization of LTBP1 and fibrillin 1 in microfibrillar structures in the ECM of cultured primary osteoblasts. Immunoelectron microscopy confirmed localization of LTBP1 to 10- to 12-nm microfibrils and suggested an ordered aggregation of LTBP1 into these structures. Early colocalization of LTBP1 with fibronectin suggested a role for fibronectin in the initial assembly of LTBP1 into the matrix; however, in more differentiated osteoblast cultures, LTBP1 and fibronectin 1 were found in distinct fibrillar networks. Overexpression of LTBP1 deletion constructs in osteoblast-like cells showed that N-terminal amino acids 67,467 were sufficient for incorporation into fibrillin-containing microfibrils and suggested that LTBP1 can be produced by cells distant from the site of fibril formation. In embryonic long bones in vivo, LTBP1 and fibrillin 1 colocalized at the surface of newly forming osteoid and bone. However, LTBP1-positive fibrils, which did not contain fibrillin 1, were present in cartilage matrix. These studies show that in addition to regulating TGF,1, LTBP1 may function as a structural component of connective tissue microfibrils. LTBP1 may therefore be a candidate gene for Marfan-related connective tissue disorders in which linkage to fibrillins has been excluded. [source]


Beer But Not Wine, Hard Liquors, or Pure Ethanol Stimulates Amylase Secretion of Rat Pancreatic Acinar Cells In Vitro

ALCOHOLISM, Issue 9 2009
Andreas Gerloff
Background:, In contrast to pure ethanol, the effect of alcoholic beverages on the exocrine pancreas is greatly unknown. Besides ethanol, alcoholic beverages contain numerous nonalcoholic constituents which might have pathophysiological effects on the pancreas. The aim of the present study was to investigate whether some commonly used alcoholic beverages and pure ethanol influence the main function of rat pancreatic acinar cells, i.e., enzyme output in vitro. Methods:, Rat pancreatic AR4-2J cells were differentiated by dexamethasone treatment for 72 hours and freshly isolated pancreatic acini were prepared from Sprague,Dawley rats using collagenase digestion. After incubation of cells in the absence or presence of 1 to 10% (v/v) beer (containing 4.7% v/v ethanol), 10% (v/v) wine (containing 10.5 to 12.5% v/v ethanol), 10% (v/v) hard liquor (such as whisky, rum, and gin), or of the corresponding ethanol concentrations (4.03 to 80.6 mM) for 60 minutes, protein secretion was measured using amylase activity assay. Results:, Incubation of AR4-2J cells with beer caused a dose-dependent stimulation of basal amylase secretion that was significant at doses of beer above 0.5% (v/v). Stimulation with 10% (v/v) beer induced 92.7 ± 25.2% of maximal amylase release in response to the most effective cholecystokinin (CCK) concentration (100 nM). In contrast, ethanol (up to 80.6 mM) did neither stimulate nor inhibit basal amylase release. Lactate dehydrogenase measurement after treatment of AR4-2J cells with beer for 24 hours indicated that the increase of amylase release was not due to cell membrane damage. Wine and hard liquor had no effect on basal amylase secretion neither diluted to the ethanol concentration of beer nor undiluted. In freshly isolated rat pancreatic acinar cells beer dose-dependently stimulated amylase secretion in a similar manner as in AR4-2J cells. Conclusions:, Our data demonstrate that beer dose-dependently increases amylase output. Since neither ethanol nor the other alcoholic beverages tested caused stimulation of amylase release, our findings indicate that nonalcoholic constituents specific for beer are responsible for this increase. These as yet unknown compounds have to be identified and considered in further studies of ethanol-induced pathological and functional changes of the pancreas. [source]


Effects of Light Exposure and Use of Intraocular Lens on Retinal Pigment Epithelial Cells In Vitro

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2009
Sheng Hui
To investigate the effect of a blue light-filtering intraocular lens (IOL) and a UV-absorbing IOL on light-induced damage to retinal pigment epithelial (RPE) cells laden with the lipofuscin fluorophore N -retinylidene- N -retinylethanolamine (A2E), A2E-laden RPE cells were exposed to white light which was filtered by either a blue light-filtering IOL or a UV-absorbing IOL. After 30 min of illumination the cell viability and the level of reactive oxygen species (ROS), free glutathione (GSH), vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) were determined. In the absence of an IOL, the white light exposure decreased cell viability to 37.2% of the nonirradiated control. The UV-absorbing IOL tended to reduce light-induced cell death; however, the decrease was not significant. The blue light-filtering IOL significantly attenuated light-induced cell damage, increasing cell viability to 79.5% of the nonirradiated control. The presence of the blue light-filtering IOL significantly increased GSH and PEDF levels, and decreased ROS and VEGF levels. This study suggests that a blue light-filtering IOL may be more protective against A2E-induced light damage and inhibit more light-induced ROS and VEGF production than a conventional UV-absorbing IOL. [source]


Oestrogenic Steroids and Melanoma Cell Interaction with Adjacent Skin Cells Influence Invasion of Melanoma Cells In Vitro

PIGMENT CELL & MELANOMA RESEARCH, Issue 2000
SHEILA MAC NEIL
The invasion of melanoma is complex and multi-staged and involves changes in both cell/extracellular matrix (ECM) and cell/cell interactions. Female steroids and ,-MSH have also been reported to influence metastatic melanoma progression, but their mechanisms of action are unknown. Accordingly, our aim was to establish in vitro models to examine (a) the influence of sex steroids and ,-melanocyte-stimulating hormone (,-MSH) on tumour invasion and the influence of (b) ECM proteins and (c) adjacent cells on melanoma invasion. In the first model, melanoma cell invasion through fibronectin over 20 hr under serum-free conditions was used to investigate the effects of 17,-oestradiol and oestrone on the invasion of human melanoma cell lines, A375-SM and HBL. A375-SM, but not HBL cells, proved very susceptible to inhibition by female steroids. However, invasion of the HBL line was inhibited by ,-MSH. Using the second model of reconstructed human skin based on de-epidermised acellular dermis, we found that the HBL cells on their own failed to invade into the dermis (irrespective of the presence or absence of the basement membrane). However, there was a significant synergistic interaction between keratinocytes, fibroblasts and HBL cells, such that a modest invasion of HBLs into the dermis was seen within 2 weeks when other skin cells were present. In contrast, A375-SM cells showed a significant ability to invade the dermis in the absence of other cells, with less invasion when other skin cells were present. In summary, these models have provided new information on the extent to which melanoma cell invasion is sensitive to oestrogenic steroids and to ,-MSH and to interaction, not only with adjacent skin cells but also to the presence of basement membrane antigens. [source]


Rapamycin-Conditioned Donor Dendritic Cells Differentiate CD4+CD25+Foxp3+ T Cells In Vitro with TGF-,1 for Islet Transplantation

AMERICAN JOURNAL OF TRANSPLANTATION, Issue 8 2010
K. L. Pothoven
Dendritic cells (DCs) conditioned with the mammalian target of rapamycin (mTOR) inhibitor rapamycin have been previously shown to expand naturally existing regulatory T cells (nTregs). This work addresses whether rapamycin-conditioned donor DCs could effectively induce CD4+CD25+Foxp3+ Tregs (iTregs) in cell cultures with alloantigen specificities, and whether such in vitro- differentiated CD4+CD25+Foxp3+ iTregs could effectively control acute rejection in allogeneic islet transplantation. We found that donor BALB/c bone marrow-derived DCs (BMDCs) pharmacologically modified by the mTOR inhibitor rapamycin had significantly enhanced ability to induce CD4+CD25+Foxp3+ iTregs of recipient origin (C57BL/6 (B6)) in vitro under Treg driving conditions compared to unmodified BMDCs. These in vitro- induced CD4+CD25+Foxp3+ iTregs exerted donor-specific suppression in vitro, and prolonged allogeneic islet graft survival in vivo in RAG,/- hosts upon coadoptive transfer with T-effector cells. The CD4+CD25+Foxp3+ iTregs expanded and preferentially maintained Foxp3 expression in the graft draining lymph nodes. Finally, the CD4+CD25+Foxp3+ iTregs were further able to induce endogenous naïve T cells to convert to CD4+CD25+Foxp3+ T cells. We conclude that rapamycin-conditioned donor BMDCs can be exploited for efficient in vitro differentiation of donor antigen-specific CD4+CD25+Foxp3+ iTregs. Such in vitro- generated donor-specific CD4+CD25+Foxp3+ iTregs are able to effectively control allogeneic islet graft rejection. [source]


Direct Stimulation of Adult Neural Stem Cells In Vitro and Neurogenesis In Vivo by Vascular Endothelial Growth Factor

BRAIN PATHOLOGY, Issue 3 2004
Anne Schänzer
Hypoxia as well as global and focal ischemia are strong activators of neurogenesis in the adult mammalian central nervous system. Here we show that the hypoxia-inducible vascular endothelial growth factor (VEGF) and its receptor VEGFR-2/Flk-1 are expressed in clonally-derived adult rat neural stem cells in vitro. VEGF stimulated the expansion of neural stem cells whereas blockade of VEGFR-2/Flk-1-kinase activity reduced neural stem cell expansion. VEGF was also infused into the lateral ventricle to study changes in neurogenesis in the ventricle wall, olfactory bulb and hippocampus. Using a low dose (2.4 ng/d) to avoid endothelial proliferation and changes in vascular permeability, VEGF stimulated adult neurogenesis in vivo. After VEGF infusion, we observed reduced apoptosis but unaltered proliferation suggesting a survival promoting effect of VEGF in neural progenitor cells. Strong expression of VEGFR-2/Flk-1 was detected in the ventricle wall adjacent to the choroid plexus, a site of significant VEGF production, which suggests a paracrine function of endogenous VEGF on neural stem cells in vivo. We propose that VEGF acts as a trophic factor for neural stem cells in vitro and for sustained neurogenesis in the adult nervous system. These findings may have implications for the pathogenesis and therapy of neurodegenerative diseases. [source]