ABSTRACT
Intervertebral disc degeneration (IDD) involves cellular changes in the nucleus pulposus (NP) characterised by a decline of the large vacuolated notochordal cells (vNCs) and a rise of smaller vacuole-free mature chondrocyte-like NP cells. An increasing number of studies demonstrate that notochordal cells (NCs) exert disease-modifying effects, establishing that NC-secreted factors are essential for the maintenance of a healthy intervertebral disc (IVD). However, understanding the role of the NCs is hampered by a restricted reserve of native cells and the lack of robust ex vivo cell model. A precise dissection enabled the isolation of NP cells from 4 d post-natal stage mouse spines and their culture into self-organised micromasses. The maintenance of cells' phenotypic characteristics was demonstrated by the presence of intracytoplasmic vacuoles and the immuno-colocalisation of the NC-markers (brachyury; SOX9) after 9 d of culture both in hypoxic and normoxic conditions. A significant increase of the size of the micromass was observed under hypoxia, consistent with a higher level of Ki-67+ immunostained proliferative cells. Furthermore, several proteins of interest for the study of vNCs phenotype (CD44; caveolin-1; aquaporin 2; patched-1) were successfully detected at the plasma membrane of NP-cells cultured in micromasses under hypoxic condition. IHC was performed on mouse IVD sections as control staining. An innovative 3D culture model of vNCs derived from mouse postnatal NP is proposed, allowing future ex vivo exploration of their basic biology and of the signalling pathways involved in IVD homeostasis that may be relevant for disc repair.
Subject(s)
Notochord , Nucleus Pulposus , Animals , Mice , Cell Membrane , Nucleus Pulposus/cytology , Notochord/cytology , Cell Hypoxia , Intervertebral Disc Degeneration/pathologyABSTRACT
Oral bisphosphonates are established therapeutics for postmenopausal osteoporosis. Alternative, simplified dosing regimens that improve tolerability and promote convenience may be advantageous. Ibandronate is a highly potent, nitrogen-containing bisphosphonate that can be administered as a convenient intravenous (i.v.) injection (over 15-30 s) in schedules featuring extended between-dose intervals. In a recent fracture prevention study, 1 and 0.5 mg i.v. ibandronate injections, given once every 3 months, were shown to dose-dependently increase lumbar spine and hip bone mineral density (BMD) and decrease biochemical markers of bone turnover in women with postmenopausal osteoporosis, but the overall magnitude of efficacy provided by both doses was suboptimal. In the present study (Intermittent Regimen intravenous Ibandronate Study: the IRIS study), the dose-response relationship with intermittent intravenous ibandronate injections was further evaluated in 520 postmenopausal osteoporotic women (aged 55-75 years, time since menopause >or= 5 years, lumbar spine [L1-L4] BMD T score < -2.5). At enrolment, participants were randomized to receive either 2 mg (n = 261) or 1 mg (n = 131) ibandronate or placebo (n = 128) intravenous injections, given once every 3 months. After 1 year, ibandronate therapy produced substantial and dose-dependent increases in lumbar spine and hip BMD, and decreases in biochemical markers of bone turnover, with the 2 mg dose providing significantly greater efficacy than the 1 mg dose. Most notably, lumbar spine BMD increased by 5.0% and 2.8% in the 2 and 1 mg groups, respectively, and decreased by 0.04% in the placebo group. Furthermore, total hip BMD increased by 2.9%, 2.2%, and 0.6%, respectively. Serum and urinary CTX, reflecting bone resorption, were decreased by 62.5% and 61%, respectively, with the 2 mg dose, and by 43.5% and 42%, respectively, with the 1 mg dose. Intravenous ibandronate was well tolerated with a similar incidence of adverse events to placebo. Importantly, no indicators of renal toxicity were reported. In summary, the 2 mg ibandronate regimen provides significantly greater BMD increases and significantly greater suppression of bone resorption markers than the 1 mg dose used in this study and in the previous fracture prevention study. Ongoing studies aim to further establish the efficacy and convenience of intermittent intravenous ibandronate injections in postmenopausal osteoporosis.
Subject(s)
Bone Resorption/drug therapy , Osteoporosis/drug therapy , Aged , Bone Density , Bone Remodeling , Diphosphonates/adverse effects , Diphosphonates/therapeutic use , Double-Blind Method , Female , Humans , Ibandronic Acid , Injections, Intravenous , Middle Aged , PlacebosABSTRACT
In order to develop an in vitro predictive assay for the detection of contact sensitizers, we investigated the possible modulation of the expression of cell-surface molecules in the early phases of treatment of murine epidermal cells (EC) with known contact sensitizers. After in vitro treatment of Balb/c EC with the strong contact sensitizer, TNBS, Langerhans cells (LCs) demonstrated a rapid up-regulation of CD45, CD40, CD32/16 (Fc gamma RII/III) and CD23 (Fc epsilon RII) molecules. CD45 and CD40 were also rapidly up-regulated on the dendritic epidermal T cells. Interestingly, after treatment with this severe sensitizer, a marked induction of CD40 expression was found on a CD45 negative population, most probably keratinocytes. In contrast to these cell-surface molecules, I-Ad/I-Ed and CD90.2 expression were unchanged. No change was observed on the expression of CD45 and CD40 after treatment with a mild or a weak contact sensitizer, citral and citronellal respectively. In contrast, like TNBS, they up-regulated the expression of CD32/16 and CD23 on LCs. The irritant sodium dodecyl sulfate had no effect on all these cell-surface molecules. Our results indicated that in vitro, chemicals with allergic potential induced early specific phenotype changes that may represent an early-activated state of the cells. This state may be responsible for initiating the afferent phase of contact sensitivity in vivo. Based on these findings, it might be possible to develop an in vitro assay to reduce the number of experimental animals for a fast screening of contact sensitizers and for discriminating between mild contact sensitizers and irritants.
Subject(s)
Allergens/pharmacology , Dermatitis, Allergic Contact/metabolism , Keratinocytes/drug effects , Langerhans Cells/drug effects , Skin/drug effects , T-Lymphocytes/drug effects , Animals , Antigens, CD/metabolism , Female , Flow Cytometry , Histocompatibility Antigens Class II/metabolism , Keratinocytes/metabolism , Langerhans Cells/metabolism , Mice , Mice, Inbred BALB C , Phenotype , Skin/metabolism , T-Lymphocytes/metabolism , Up-RegulationABSTRACT
For the development of mechanistic assays in immunotoxicology, the phenotype, cytokine production, and stimulatory function of dendritic cells (DCs) were assessed after incubation with the chemical haptens aminophenol, chlorpromazine hydrochloride, dinitrochlorobenzene (DNCB), and with the DNCB-corresponding tolerogen DCNB, the metal allergen nickel sulfate, the irritants sodium dodecyl sulfate and benzoic acid, as well as with staphylococcal enterotoxin B (SEB) and lipopolysaccharide (LPS). DCs were differentiated from human monocytes by in vitro exposure to GM-CSF and interleukin-4 (IL-4) for 7 days. Flow cytometric data revealed that only representative haptens increased the surface expression of HLA-DR, CD86, CD40, and of CD54 on DCs when compared to irritants or to the tolerogen. This event was associated with an increased ability of DCs to stimulate T cell proliferation. Moreover, after incubation with the haptens, but not with the irritants or the tolerogen, a higher production of TNF-alpha by DCs was observed. Under our experimental conditions, no release of IL-1beta, IL-10, or IL-12 was detected. Compared to the activation elicited by haptens, SEB strongly up-regulated HLA-DR and costimulatory molecule expression. In agreement with this effect, there was a marked release of TNF-alpha and a slight production of IL-12. IL-1beta and IL-10 were not detected in the culture medium. Finally, SEB-pulsed DCs showed a strong T-cell-stimulating activity. These data underline the activating potential of haptens versus irritants or a tolerogen on DC functions. The different levels of DC activation by haptens and SEB suggested that distinct cellular events were involved.
Subject(s)
Allergens/toxicity , Dendrites/drug effects , Haptens/toxicity , Metals/toxicity , Monocytes/drug effects , Staphylococcus aureus/immunology , Superantigens/toxicity , Biomarkers , Cells, Cultured , Cytokines/metabolism , Dendrites/metabolism , Dendrites/ultrastructure , Enterotoxins/pharmacology , Flow Cytometry , HLA-DR Antigens/immunology , Humans , Lipopolysaccharides/pharmacology , Monocytes/metabolism , Monocytes/ultrastructure , Phenotype , T-Lymphocytes/immunologyABSTRACT
We analyzed the effect of isoprenoid depletion by fluvastatin on bradykinin (BK)- and epidermal growth factor (EGF)-mediated Ca2+ mobilization and prostaglandin E2 production, in the human keratinocyte cell line HaCaT. BK and EGF stimulated Ca2+ mobilization in an agonist-dependent manner. The synthesis of prostaglandin E2 paralleled the level of Ca2+ mobilization induced by BK and EGF. Treatment with fluvastatin increased the EGF-promoted but not the BK-promoted Ca2+ mobilization and prostaglandin E2 production in Ca(2+)-containing medium. In the absence of extracellular Ca2+, fluvastatin treatment led to an increase in intracellular Ca2+ release by both agonists. This effect was abolished by depleting the intracellular pool of Ca2+ with thapsigargin. Our findings showed that the intracellular Ca2+ release was dependent on the metabolism of mevalonate and that the Ca2+ mobilization modulated prostaglandin E2 synthesis in human keratinocytes.
Subject(s)
Calcium/metabolism , Fatty Acids, Monounsaturated/pharmacology , Indoles/pharmacology , Keratinocytes/drug effects , Aniline Compounds/metabolism , Bradykinin/pharmacology , Cell Line , Dinoprostone/biosynthesis , Epidermal Growth Factor/pharmacology , Fluvastatin , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Kinetics , Mevalonic Acid/metabolism , Thapsigargin/pharmacology , Xanthenes/metabolismABSTRACT
In HaCaT keratinocytes bradykinin-triggered actin reorganization was inhibited by quinacrine, a phospholipase A2 inhibitor, and restored by addition of arachidonic acid. Bradykinin-induced actin breakdown and cortical actin formation were respectively prevented by indomethacin, a cyclooxygenase inhibitor, and nordihydroguaiaretic acid, a lipoxygenase inhibitor. Addition of prostaglandins or leukotrienes, respectively, reversed the effects of inhibitors. This suggested a crucial role for a cyclooxygenase product in actin depolymerization and for a lipoxygenase product in cortical actin formation. Furthermore, we found that bradykinin stimulated HaCaT keratinocyte migration. This event was blocked by quinacrine, indomethacin or nordihydroguaiaretic acid, and restored by addition of prostaglandins or leukotrienes, respectively. We also showed that genistein, a tyrosine kinase inhibitor, inhibited HaCaT cell locomotion. In conclusion, bradykinin modulated actin reorganization and cell motility in keratinocytes, probably by a mechanism involving arachidonic acid metabolites and a tyrosine kinase activity.
Subject(s)
Actins/metabolism , Bradykinin/physiology , Cell Movement/physiology , Keratinocytes/cytology , Cell Line, Transformed , Enzyme Inhibitors/pharmacology , Humans , Indomethacin/pharmacology , Keratinocytes/drug effects , Masoprocol/pharmacology , Phospholipases A/antagonists & inhibitors , Phospholipases A2 , Quinacrine/pharmacologyABSTRACT
We investigated the effect of bradykinin on the expression of the proto-oncogenes c-fos, c-jun and c-myc and on cell proliferation in the human keratinocyte cell line, HaCaT. Analysis of mRNAs was done by Northern blotting with single-stranded DNA hybridization probes. Bradykinin caused a rapid and transient accumulation in c-fos and c-jun mRNAs. In contrast, c-myc mRNA increased more slowly. Moreover, we report that bradykinin was a weak stimulator of HaCaT cell proliferation whereas epidermal growth factor, which induced the same degree of mRNA elevation, was shown as a powerful mitogen. Thus, while in HaCaT cells bradykinin promotes the expression of the proto-oncogenes c-fos, c-jun and c-myc, other biochemical events appear to be necessary for cell division.
Subject(s)
Bradykinin/pharmacology , Gene Expression Regulation/drug effects , Genes, Immediate-Early/drug effects , Keratinocytes/drug effects , Proto-Oncogenes/drug effects , RNA, Messenger/analysis , Base Sequence , Cells, Cultured , Humans , Keratinocytes/metabolism , Molecular Sequence DataABSTRACT
Bradykinin is an inflammatory mediator which activates signalling pathways in human keratinocytes via a receptor linked to a GTP-binding protein. In the HaCaT human keratinocyte cell line, we observed bradykinin-stimulated tyrosine phosphorylation of several cellular proteins with peak response at 15 min. The focal adhesion proteins paxillin and p125FAK were tyrosine phosphorylated in response to bradykinin but not in response to epidermal growth factor. Interestingly, we identified the epidermal growth factor receptor as a novel target for bradykinin-induced tyrosine phosphorylation. The tyrosine kinase inhibitor genistein and the protein kinase C inhibitors staurosporine and Ro31-7549, all blocked bradykinin-induced tyrosine phosphorylation. Our data suggest that stimulation of the bradykinin receptor leads to activation of a tyrosine kinase activity via a protein-kinase-C-dependent pathway in human keratinocytes.
Subject(s)
Bradykinin/pharmacology , Cell Adhesion Molecules/metabolism , Cytoskeletal Proteins/metabolism , ErbB Receptors/metabolism , Keratinocytes/metabolism , Phosphoproteins/metabolism , Protein-Tyrosine Kinases/metabolism , Alkaloids/pharmacology , Cell Adhesion Molecules/isolation & purification , Cell Line , Chromatography, Affinity , Cytoskeletal Proteins/isolation & purification , Electrophoresis, Polyacrylamide Gel , ErbB Receptors/isolation & purification , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Genistein , Humans , Immunoblotting , Indoles/pharmacology , Isoflavones/pharmacology , Keratinocytes/drug effects , Maleimides/pharmacology , Molecular Weight , Paxillin , Phosphoproteins/isolation & purification , Phosphotyrosine , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/isolation & purification , Receptor, Insulin/metabolism , Receptors, Bradykinin/physiology , Staurosporine , Tyrosine/analogs & derivatives , Tyrosine/analysis , Tyrosine/metabolismABSTRACT
Histochemical and ultrastructural studies have already demonstrated that chronic exposure to UV radiation induces profound alterations in all structural elements of the skin and that topical all-trans retinoic acid (tRA) can substantially correct much of the tissue damage. However, previous biochemical studies on dermal components of the extracellular matrix have led to contradictory results, particularly with regard to the effect of chronic UV exposure. The aim of our study was to investigate changes in collagen content and other dermal modifications induced by tRA in irradiated and non-irradiated hairless mouse skin. Hairless mice were exposed to increasing doses of UVB for 10 weeks (the cumulative total dose was 4.6 J cm-2). After the UV irradiation period the animals were treated with 0.05% tRA or with ethanol-polyethylene glycol vehicle alone three times a week for up to 10 weeks. Non-irradiated animals underwent the same treatments. The main clinical and histological changes induced by UVB exposure were erythema, wrinkling, keratosis and epidermal thickening. Following UVB exposure, tRA treatment did not improve the clinical aspect but increased the width of the dermal repair zone. Fibronectin, laminin and type I and VI collagens were detected by indirect immunofluorescence techniques in this zone. Type I and III collagens were quantitated in skin fragments after cyanogen bromide digestion and polyacrylamide gel electrophoresis. Under our experimental conditions, UVB irradiation alone induced neither changes in total collagen nor in type I and III collagen levels. tRA treatment of irradiated skin significantly increased both type I and III collagen levels by factors of 1.33 and 1.88 respectively. The ratio of type III to types I + III increased significantly. Topical tRA also increased collagen type levels in non-irradiated hairless mouse skin. Type I collagen increased proportionally to type III. This study leads to the conclusion that topical tRA exerts direct or indirect effects on collagen metabolism in irradiated as well as non-irradiated hairless mouse skin.