High doses of dexamethasone induce increased beta-cell proliferation in pancreatic rat islets

Rafacho A, Cestari TM, Taboga SR, Boschero AC, Bosqueiro JR. High doses of dexamethasone induce increased beta-cell proliferation in pancreatic rat islets. Am J Physiol Endocrinol Metab 296: E681-E689, 2009. First published January 21, 2009; doi:10.1152/ajpendo.90931.2008.-Activation of insulin signaling and cell cycle intermediates is required for adult beta-cell proliferation. Here, we report a model to study beta-cell proliferation in living rats by administering three different doses of dexamethasone (0.1, 0.5, and 1.0 mg/kg ip, DEX 0.1, DEX 0.5, and DEX 1.0, respectively) for 5 days. Insulin sensitivity, insulin secretion, and histomorphometric data were investigated. Western blotting was used to analyze the levels of proteins related to the control of beta-cell growth. DEX 1.0 rats, which present moderate hyperglycemia and marked hyperinsulinemia, exhibited a 5.1-fold increase in beta-cell proliferation and an increase (17%) in beta-cell size, with significant increase in beta-cell mass, compared with control rats. The hyperinsulinemic but euglycemic DEX 0.5 rats also showed a significant 3.6-fold increase in beta-cell proliferation. However, DEX 0.1 rats, which exhibited the lowest degree of insulin resistance, compensate for insulin demand by improving only islet function. Activation of the insulin receptor substrate 2/phosphatidylinositol 3-kinase/serine-threoninekinase/ribosomalprotein S6 kinase pathway, as well as protein retinoblastoma in islets from DEX 1.0 and DEX 0.5, but not in DEX 0.1, rats was also observed. Therefore, increasing doses of dexamethasone induce three different degrees of insulin requirement in living rats, serving as a model to investigate compensatory beta-cell alterations. Augmented beta-cell mass involves beta-cell hyperplasia and, to a lower extent, beta-cell hypertrophy. We suggest that alterations in circulating insulin and, to a lesser extent, glucose levels could be the major stimuli for beta-cell proliferation in the dexamethasone-induced insulin resistance.

Bone cell responses to the composite of Ricinus communis polyurethane and alkaline phosphatase

The aim of this study was to evaluate the response of osteoblastic cells to the composite of Ricinus cominunis polyurethane (RCP) and alkaline phosphatase (ALP) incubated in synthetic body fluid (SBF). RCP pure (RCPp) and RCP blended with ALP 6 mg/mL polymer (RCP+ALP) were incubated in SBF for 17 days. Four groups of RCP were tested: RCPp, RCP+ALP, and RCPp and RCP+ALP incubated in SBF (RCPp/SBF and RCP+ALP/SBF). Stem cells from rat bone marrow were cultured in conditions that allowed osteoblastic differentiation on RCP discs and were evaluated: cell adhesion, culture growth, cell viability, total protein content, ALP activity, and bone-like nodule formation. Data were compared by ANOVA or Kruskal-Wallis test. The group RCP-A P was highly cytotoxic and, therefore, was not considered here. Cell adhesion (p = 0.14), culture growth (p = 0.39), viability (p = 0.46) and total protein content (p = 0.12) were not affected by either RCP composition or incubation in SBE ALP activity was affected (p = 0.0001) as follows: RCPp < RCPp/SBF < RCP+ALP/SBF. Bone-like nodule formation was not observed on all evaluated groups. The composite RCP+ALP prior to SBF incubation is cytotoxic and must not be considered as biomaterial, but the incorporation of ALP to the RCP followed by SBF incubation could be a useful alternative to improve the biological properties of the RCP. (c) 2007 Wiley Periodicals, Inc.

In vitro osteogenesis induced by cells derived from sites submitted to sinus grafting with anorganic bovine bone

Objectives: This study evaluated key parameters of the in vitro osteogenesis induced by osteoblastic cells obtained from sites submitted to sinus grafting with anorganic bovine bone (ABB) in comparison with cells derived from bone sites of the same patients. Materials and methods: In three patients, the augmentation of maxillary sinus was carried out using ABB (Bio-Oss (R)). After at least 6 months, during the surgical intervention for titanium implants placement, biopsies were taken from these areas using trephine burs (grafted group). Bone fragments, of the same patients, from sites that had not received graft were also obtained with trephine burs and used as a control group. Osteoblastic cells were obtained from grafted and control groups by enzymatic digestion and cultured under standard osteogenic condition until subconfluence. First passaged cells were cultured in 24-well culture plates. Cell adhesion was evaluated at 24 h. For proliferation and viability assay, cells were cultured for 1, 3, 7, and 10 days. Total protein content and alkaline phosphatase (ALP) activity were measured at 3, 7, 10, 14, 17, and 21 days. Cultures were stained with Alizarin red S at 21 days, for detection of mineralized matrix. Data were compared by Student`s t-test. Results: Cell adhesion and viability were not affected by cell source (P>0.05). Total protein content was greater (P<0.05) for grafted group. Cell proliferation, ALP activity, and bone-like nodule formation were all greater (P<0.05) for the control group. Conclusions: Taken together, these results indicate that the in vivo long-term contact of cells with ABB downregulates the expression of osteoblast phenotype and consequently the in vitro osteogenesis.

Increased circulating levels of matrix metalloproteinase (MMP)-8, MMP-9, and pro-inflammatory markers in patients with metabolic syndrome

Background: Metabolic syndrome (MetS) predisposes to cardiovascular complications. Increased concentrations of pro-inflammatory mediators and imbalanced concentrations of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) may reflect the pathophysiology of MetS. We compared the circulating levels of MMPs, TIMPs, and inflammatory mediators in MetS patients with those found in healthy controls. Methods: We studied 25 healthy subjects and 25 MetS patients. The plasma levels of pro-MMP-2 and pro-MMP-9 were determined by gelatin zymography. The plasma concentrations of MMP-8, MMP-3, TIMP-1, TIMP-2, monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), intercellular adhesion molecule (sICAM-1), and sP-selectin were measured by ELISA kits. Results: We found higher sP-selectin, sICAM-1, MCP-1, and IL-6 (all P<0.05) concentrations in MetS patients compared with healthy controls. No differences in pro-MMP-2, MMP-3, and TIMP-2 levels were found (all P>0.05). However, we found higher pro-MMP-9, MMP-8. and TIMP-1 levels in MetS patients compared with healthy controls (all P<0.05). Conclusions: Patients with MetS have increased circulating concentrations of pro-MMP-9, MMP-8, and TIMP-1 that are associated with increased concentrations of pro-inflammatory mediators and adhesion molecules. These findings suggest that MMPs may have a role in the increased cardiovascular risk of MetS patients. Pharmacological interventions targeting MMPs, especially MMP-9 and MMP-8 deserve further investigation in MetS patients. (C) 2009 Elsevier B.V. All rights reserved.

Human alveolar bone cell proliferation, expression of osteoblastic phenotype, and matrix mineralization on porous titanium produced by powder metallurgy

Objective: This study aimed at investigating the influence of the porous titanium (Ti) structure on the osteogenic cell behaviour. Materials and methods: Porous Ti discs were fabricated by the powder metallurgy process with the pore size typically between 50 and 400 mm and a porosity of 60%. Osteogenic cells obtained from human alveolar bone were cultured until subconfluence and subcultured on dense Ti (control) and porous Ti for periods of up to 17 days. Results: Cultures grown on porous Ti exhibited increased cell proliferation and total protein content, and lower levels of alkaline phosphatase (ALP) activity than on dense Ti. In general, gene expression of osteoblastic markers-runt-related transcription factor 2, collagen type I, alkaline phosphatase, bone morphogenetic protein-7, and osteocalcin was lower at day 7 and higher at day 17 in cultures grown on porous Ti compared with dense Ti, a finding consistent with the enhanced growth rate for such cultures. The amount of mineralized matrix was greater on porous Ti compared with the dense one. Conclusion: These results indicate that the porous Ti is an appropriate substrate for osteogenic cell adhesion, proliferation, and production of a mineralized matrix. Because of the three-dimensional environment it provides, porous Ti should be considered an advantageous substrate for promoting desirable implant surface-bone interactions.

Human Alveolar Bone-Derived Cell-Culture Behaviour on Biodegradable Poly(L-lactic Acid)

Poly(L-lactic acid) (PLA) is a polymer of great technological interest, whose excellent mechanical properties, thermal plasticity and bioresorbability render it potentially useful for environmental applications, as a biodegradable plastic and as a biocompatible material in biomedicine. The interactions between an implant material surface and host cells play central roles in the integration, biological performance and clinical success of implanted biomedical devices. Osteoblasts from human alveolar bone were chosen to investigate the cell behaviour when in contact with PLA discs. Cell morphology and adhesion through osteopontin (OPN) and fibronectin (FN) expression were evaluated in the initial osteogenesis, as well as cell proliferation, alkaline phosphatase activity and bone nodule formation. It was shown that the polymer favoured cell attachment. Cell proliferation increased until 21 days but in a smaller rate when compared to the control group. On the other hand, ALP activity and bone mineralization were not enhanced by the polymer. It is suggested that this polymer favours cell adhesion in the early osteogenesis in vitro, but it does not enhance differentiation and mineralization. (C) Koninklijke Brill NV, Leiden, 2009

Stromal-derived factor-1 alpha (CXCL12) levels increase in periodontal disease

Background: The CXC chemokine receptor 4 (CXCR4) and its ligand, stromal cell-derived factor-1 (SDF-1 alpha or CXC chemokine ligand 12) are involved in the trafficking of leukocytes into and out of extravascular tissues. The purpose of this study was to determine whether SDF-1 alpha secreted by host cells plays a role in recruiting inflammatory cells into the periodontia during local inflammation. Methods: SDF-1 alpha levels were determined by enzyme-linked immunosorbent assay in gingival crevicular fluid (GCF) of 24 individuals with periodontitis versus healthy individuals in tissue biopsies and in a preclinical rat model of Porphyromonas gingivalis lipopolysaccharide-induced experimental bone loss. Neutrophil chemotaxis assays were also used to evaluate whether SDF-1 alpha plays a role in the recruitment of host cells at periodontal lesions. Results: Subjects with periodontal disease had higher levels of SDF-1 alpha in their GCF compared to healthy subjects. Subjects with periodontal disease who underwent mechanical therapy demonstrated decreased levels of SDF-1 alpha. Immunohistologic staining showed that SDF-1 alpha and CXCR4 levels were elevated in samples obtained from periodontally compromised individuals. Similar results were observed in the rodent model. Neutrophil migration was enhanced in the presence of SDF-1 alpha, mimicking immune cell migration in periodontal lesions. Conclusions: SDF-1 alpha may be involved in the immune defense pathway activated during periodontal disease. Upon the development of diseased tissues, SDF-1 alpha levels increase and may recruit host defensive cells into sites of inflammation. These studies suggest that SDF-1 alpha may be a useful biomarker for the identification of periodontal disease progression.

In vitro osteogenesis on fluorcanasite glass-ceramic with three different chemical compositions

This study aimed at investigating in vitro osteogenesis on three fluorcanasite glass-ceramic compositions with different solubilities (K3, K5, and K8). Osteoblastic cells were obtained from human alveolar bone fragments and cultured under standard osteogenic condition until subconfluence. First passage cells were cultured on K3, K5, and K8 and on Bioglass (R) 45S5 (45S5-control). Cell adhesion was evaluated at 24 h. For proliferation and viability, cells were cultured for 1, 4, and 10 days. Total protein content and alkaline phosphatase (ALP) activity were measured at 7, 14, and 21 days. Cultures were stained with Alizarin red at 21 days, for detection of mineralized matrix. Data were compared by ANOVA followed by Duncan`s test. Cell adhesion, cell proliferation, viability, total protein content, and ALP activity were not affected by fluorcanasite glass-ceramic composition and solubility. Bone-like formation was similar on all fluorcanasite-glass ceramics and was reduced compared to 45S5. The changes in the chemical composition and consequently solubility of the fluorcanasite glass-ceramics tested here did not significantly alter the in vitro osteogenesis. Further modifications of the chemical composition of the fluorcanasite glass-ceramic would be required to improve bone response, making this biomaterial a good candidate to be employed as a bone substitute.

Cramoll 1,4 lectin increases ROS production, calcium levels, and cytokine expression in treated spleen cells of rats

This study reports the in vivo stimulatory effects of Cramoll 1,4 on rat spleen lymphocytes as evidenced by an increase in intracellular reactive oxygen species (ROS) production, Ca(2+) levels, and interleukin (IL)-1 beta expression. Cramoll 1,4 extracted from seeds of the Leguminosae Cratylia mollis Mart., is a lectin with antitumor and lymphocyte mitogenic activities. Animals (Nine-week-old male albino Wistar rats, Rattus norvegicus) were treated with intraperitoneal injection of Cramoll 1,4 (235 mu g ml(-1) single dose) and, 7 days later, spleen lymphocytes were isolated and analyzed for intracellular ROS, cytosolic Ca(2+), and IL-6, IL-10, and IL-1 mRNAs. Cell viability was investigated by annexin V-FITC and 7-amino-actinomycin D staining. The data showed that in lymphocytes activated by Cramoll 1,4 the increase in cytosolic and mitochondrial ROS was related to higher cytosolic Ca(2+) levels. Apoptosis and necrosis were not detected in statistically significant values and thus the lectin effector activities did not induce lymphocyte death. In vivo Cramoll 1,4 treatment led to a significant increase in IL-1 beta but IL-6 and -10 levels did not change. Cramoll 1,4 had modulator activities on spleen lymphocytes and stimulated the Th2 response.

Deficiency of the Cytoskeletal Protein SPECC1L Leads to Oblique Facial Clefting

Genetic mutations responsible for oblique facial clefts (ObFC), a unique class of facial malformations, are largely unknown. We show that loss-of-function mutations in SPECC1L. are pathogenic for this human developmental disorder and that SPECC1L is a critical organizer of vertebrate facial morphogenesis. During murine embryogenesis, Speed 1 1 is expressed in cell populations of the developing facial primordial, which proliferate and fuse to form the face. In zebrafish, knockdown of a SPECC1L homolog produces a faceless phenotype with loss of jaw and facial structures, and knockdown in Drosophila phenocopies mutants in the integrin signaling pathway that exhibit cell-migration and -adhesion defects. Furthermore, in mammalian cells, SPECC1L colocalizes with both tubulin and actin, and its deficiency results in defective actin-cytoskeleton reorganization, as well as abnormal cell adhesion and migration. Collectively, these data demonstrate that SPECC1L functions in actin-cytoskeleton reorganization and is required for proper facial morphogenesis.

The effect of DMSA-functionalized magnetic nanoparticles on transendothelial migration of monocytes in the murine lung via a beta(2) integrin-dependent pathway

Magnetic nanoparticles surface-functionalized with meso-2,3-dimercaptosuccinic acid (MNPs-DMSA) constitute an innovative and promising approach for tissue- and cell-targeted delivery of therapeutic drugs in the lung. Transendothelial migration of leukocytes in the lung is a side effect of endovenous administration of MNPs-DMSA. Using cytologic and phenotypic analysis of murine bronchoalveolar lavage cells, we identified monocytes/macrophages as the main subpopulation of leukocytes involved in this process. Moreover, ultrastructural analysis revealed the presence of nanoparticles inside of numerous macrophages from bronchoalveolar lavage. MNPs-DMSA at concentrations as high as 1 X 10(15) nanoparticles/mL had no toxic effects on macrophages, as evidenced by 3-(4, 5-dimethylthiazolyi-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Notably, MNPs-DMSA up-regulated the mRNA expression of E, L- and P-selectin and macrophage-1 antigen in the murine lung. Upregulation of these cell adhesion molecules was associated with an increased concentration of tumor necrosis factor-alpha in lung. Finally, the critical relevance of the beta(2) integrin-dependent pathway in leukocyte transmigration elicited by MNPs-DMSA was demonstrated by use of knockout mice. Our results characterize mechanisms of the pro-inflammatory effects of MNPs-DMSA in the lung, and identify beta(2) integrin-targeted interventions as promising strategies to reduce pulmonary side effects of MNPs-DMSA during biomedical applications. (C) 2009 Elsevier Ltd. All rights reserved.

Amlodipine reduces the antimigratory effect of diclofenac in spontaneously hypertensive rats

Amlodipine, an antihypertensive drug, and diclofenac, an anti inflammatory drug, may generally be combined, particularly in elderly patients; therefore, the potential for their interaction is high. We aim to determine if amlodipine interferes with the antimigratory effect of diclofenac. For this, male spontaneously hypertensive rats (SHRs) were treated with either diclofenac (1 mg.kg(-1).d(-1), 15 d) alone or combined with amlodipine (10 mg.kg(-1).d(-1), 15 d). Leukocyte rolling, adherence, and migration were studied by intravital microscopy. Diclofenac did not change (180.0 +/- 2.3), whereas amlodipine combined (163.4 +/- 5.1) or not (156.3 +/- 4.3) with diclofienac reduced the blood pressure (BP) levels in SHR (183.1 +/- 4.4). Diclofenac and amlodipine reduced leukocyte adherence, migration, and ICAM-I expression, whereas only diclofenac reduced rolling leukocytes as well. Combined with amlodipine, the effect of the diclofenac was reduced. Neither treatment tested increased the venular shear rate or modified the venular diameters, number of circulating leukocytes, P-selectin, PECAM-1, L-selectin, or CD-18 expressions. No difference could be found in plasma concentrations of both drugs given alone or in association. In conclusion, amlodipine reduces leukocyte migration in SHR, reducing endothelial cell ICAM-1 expression. Amlodipine reduces the effect of the diclofenac, possibly by the same mechanism. A pharmacokinetic interaction as well as an effect on the other adhesion molecules tested could be discarded.

PAS-1, an Ascaris suum Protein, Modulates Allergic Airway Inflammation via CD8(+) gamma delta TCR(+) and CD4(+) CD25(+) FoxP3(+) T Cells

We have previously demonstrated that PAS-1, a 200 kDa protein from Ascaris suum, has a potent immunomodulatory effect on humoral and cell-mediated responses induced by APAS-3 (an allergenic protein from A. suum) or unrelated antigens. In this study, we investigated the mechanisms by which PAS-1 is able to induce this effect on an allergic airway inflammation induced by OVA in mice. C57BL/6 mice were adoptively transferred on day 0 with seven different PAS-1-primed cell populations: PAS-1-primed CD19(+) or B220(+) or CD3(+) or CD4(+) or CD8(+) or CD4(+) CD25) or CD4(+) CD25(+) lymphocytes. These mice were immunized twice with OVA and alum by intraperitoneal route (days 0 and 7) and challenged twice by intranasal route (days 14 and 21). Two days after the last challenge, the airway inflammation was evaluated by antibody levels, cellular migration, eosinophil peroxidase levels, cytokine and eotaxin production, and pulmonary mechanical parameters. Among the adoptively transferred primed lymphocytes, only CD4(+) CD25(+), CD8(+) or the combination of both T cells impaired the production of total IgE and OVA-specific IgE and IgG1 antibodies, eosinophilic airway inflammation, Th2-type cytokines (IL-4, IL-5 and IL-13), eotaxin release and airway hyperreactivity. Moreover, airway recruited cells from CD4(+) CD25(+) and CD8(+) T-cell recipient secreted more IL-10/TGF-beta and IFN-gamma, respectively. Moreover, we found that PAS-1 expands significantly the number of CD4(+) CD25(+) FoxP3(+) and CD8(+) gamma delta TCR(+) cells. In conclusion, these findings demonstrate that the immunomodulatory effect of PAS-1 is mediated by these T-cell subsets.

Structural effects and hydrogen bonds on N,N `-di(methoxycarbonylsulfenyl)urea, [CH(3)OC(O)SNH](2)CO, studied by experimental and theoretical methods

Pure N,N`-di(methoxycarbonylsulfenyl)urea, [CH(3)OC(O)SNH](2)CO, is quantitatively prepared by the hydrolysis reaction of CH(3)OC(O)SNCO and characterized by (1)H NMR, GC-MS and FTIR spectroscopy techniques. Structural and conformational properties are analyzed using a combined approach with data obtained from X-ray diffraction, vibrational spectra and theoretical calculation methods. The IR and Raman spectra for normal and deuterated species are reported. The crystal structure of [CH(3)OC(O)SNH](2)CO was determined by X-ray diffraction methods. The substance crystallizes in the orthorhombic P2(1)2(1)2 space group with a = 9.524(2), b = 12.003(1), c = 4.481 (1) angstrom, and Z = 2 moieties in the unit cell. The molecule is sited on a twofold crystallographic axis (C(2)) parallel to c and shows the anti-anti conformation (S-N single bonds antiperiplanar with respect to the opposite C-N single bonds in sulfenyl-urea-sic group). Neighboring molecules are arranged in a chain motif that extends along the C(2)-axis and is held by bifurcated NH center dot center dot center dot O center dot center dot center dot HN intermolecular bonds. A local planar symmetry is observed in the crystal for the central -SN(H)C(O)N(H)S- skeleton. Experimental and calculated data allow to trace this structural feature to the occurrence of N-H center dot center dot center dot O=C hydrogen bonding interactions. Calculated vibrational and structural properties are in good agreement with the experimentally determined features. (C) 2008 Elsevier B.V. All rights reserved.

Glypican-3 regulates migration, adhesion and actin cytoskeleton organization in mammary tumor cells through Wnt signaling modulation

Glypican-3 (GPC3) is a proteoglycan involved in migration, proliferation and cell survival modulation in several tissues. There are many reports demonstrating a downregulation of GPC3 expression in some human tumors, including mesothelioma, ovarian and breast cancer. Previously, we determined that GPC3 reexpression in the murine mammary adenocarcinoma LM3 cells induced an impairment of their in vivo invasive and metastatic capacities together with a higher susceptibility to in vitro apoptosis. Currently, the signaling mechanism of GPC3 is not clear. First, it was speculated that GPC3 regulates the insulin-like growth factor (IGF) signaling system. This hypothesis, however, has been strongly challenged. Recently, several reports indicated that at least in some cell types GPC3 serves as a selective regulator of Wnt signaling. Here we provide new data demonstrating that GPC3 regulates Wnt pathway in the metastatic adenocarcinoma mammary LM3 cell line. We found that GPC3 is able to inhibit canonical Wnt signals involved in cell proliferation and survival, as well as it is able to activate non canonical pathway, which directs cell morphology and migration. This is the first report indicating that breast tumor cell malignant properties can be reverted, at least in part, by GPC3 modulation of Wnt signaling. Our results are consistent with the potential role of GPC3 as a metastasis suppressor.