76 research outputs found
Models for investigating functional roles of osteopontin: characterization of anti-OPN monoclonal antibodies
No full textOsteopontin is an integrin-binding phosphorylated glycoprotein. It is found in many tissues and can be found in all body fluids. It is associated with several cellular processes such as cell survival, cancer progression and stress response. These particular cellular processes were of interest in my research.
This thesis is divided into two parts. Part I being about the purification of OPN and characterization of anti-OPN monoclonal antibodies. In order to accomplish part I of this thesis, OPN was purified by ion-exchange and desalting chromatography, and analyzed by SDS-PAGE followed by Coomassie Blue staining. The methods for purifying anti-OPN mAbs from hybridomas by ascites consisted of ammonium sulfate precipitation, ion-exchange chromatography, and protein A/G purification. Anti-OPN mAbs were characterized by SDS-PAGE analysis, Western blotting and ELISAs. The results from these analyses were then compiled into our database. The OPN and mAbs that were made were used in several experiments by my fellow lab mates and me.
The second part of my thesis was to investigate potential models for characterizing the functional roles of OPN. This was accomplished by studying cell survival with human umbilical vein endothelial cells (HUVECs) and cancer cell phenotypes with the pancreatic cancer cell line HS766T. Flow cytometry was used to analyze cell survival in HUVECs. Transfection with specific plasmids designed to increase or decrease OPN expression was used to modulate the cancer cell phenotype of HS766T cells.
In conclusion, I was able to successfully generate several pure stable preparations of OPN and anti-OPN mAbs. Also, I was able to reproduce cell survival in HUVECS by OPN a few times but because it was not consistently reproducible, I was unable to tests the effects of mAbs on the survival of HUVECs by OPN. Also, since our research was already successfully carried out and published by another group, I decided not to purse this project further. Lastly, I was able to create several clones of HS766T that exhibited increased or decreased OPN expression as determined by Real-Time PCR analysis.M.S.Includes bibliographical references (p. 55-60)by Dana M. Cifell
OPN-a Splicing Variant Expression in Non-small Cell Lung Cancer and its Effects on the Bone Metastatic Abilities of Lung Cancer Cells In Vitro
No full textBackground: Osteopontin (OPN) is known to be involved in the development of certain cancers, including non-small cell lung cancer (NSCLC). However, its role in tumour progression remains unclear. The present study investigated the expression and biological impact of the OPN variant, OPN-a in NSCLC. Materials and Methods: OPN-a splicing variant expression in human NSCLC tissues was analyzed by real-time qPCR and immunohistochemistry (IHC), respectively. The impact of OPN-a on cellular functions of lung cancer cells was also evaluated. In addition, an in vitro model was developed for the assessment of interactions between lung cancer cells and bone tissue. Results: The expression of OPN-a was higher in lung cancer tissues compared to normal controls. OPN-a promoted the malignant phenotypes of A549 cells by enhancing cell-adherent abilities to bone tissues, which could be mediated by the interaction with the cell surface receptor ava3 integrin. Conclusion: OPN-a may represent a bone metastatic factor in human lung cancer, as well as a potential therapy target.National Natural Science Foundation of the People's Republic of China [81572704]; Cancer Research Wales; Albert Hung Foundation; Cardiff University China Medical ScholarshipSCI(E)ARTICLE52245-22543
Expression and function of osteopontin variants in HCV-related liver disease and hepatocellular carcinoma.
No full textOsteopontin (OPN) is a highly secreted multi-functional sialoprotein that is widely expressed in tissues, blood and urine. It is involved in a number of normal physiological functions, but is also significantly elevated in a number of cancers. While OPN is significantly expressed in hepatocellular carcinoma (HCC) little is known as to its role and if it is expressed in the pre-cancerous hepatitis C virus (HCV) infected liver. In this thesis we show that OPN is expressed in the liver and in HCC as three variants, the full-length protein OPN-A and two splice variants OPN-B and OPN-C. Through production of stable Huh-7 cells expressing the OPN variants, we show for the first time that all variants increase proliferation of a range of cultured hepatoma cell lines in a paracrine manner through
interactions with the cell surface OPN receptor CD44. Similarly, OPN-A (and to a lesser extent OPN-B and –C) accelerated Huh-7 derived tumor growth in a nude mouse model. We also show for the first time expression of all three OPN variants in the non-diseased liver as it was previously thought that splicing was a feature specific for tumor cells. Clinically, OPN is known to be highly expressed in HCC, however, its expression in chronic hepatitis C is not well documented. In this thesis we show that OPN mRNA expression is elevated in the HCV-infected liver with a trend towards increased expression as liver disease progresses. Consistent with an increase in mRNA, serum OPN levels were also increased in the HCV-infected liver although we could find no correlation with degree of liver disease. However, our sample size was small and this section of the thesis needs repeating with a larger HCV-infected patient cohort. Furthermore, we show that elevated OPN expression is not specific to the HCV-infected liver as OPN is also elevated in the HBV-infected and alcoholic liver suggesting that HCV does not drive OPN expression but is more likely as a result of the inflammatory process in the viral infected liver. Interestingly we also show that there is a shift of OPN expression from bile duct epithelial cells in the non-diseased liver to the hepatocyte in the HCV-infected liver which raises the question as to the role of OPN in hepatocyte transformation to facilitate the development of HCC. Our evaluation of serum OPN expression also suggests that OPN has potential as both a diagnostic and potentially prognostic biomarker for not only HCC (arising from HBV and HCV infections and alcohol abuse) but also the earlier stages of HCV-related liver disease. This work for the first time characterises the expression of all OPN variants in the liver including HCC and may be useful for identifying targeted OPN-based therapeutic approaches for HCC and other cancers. Furthermore it also suggests that monitoring OPN in chronic hepatitis C may be
useful in monitoring liver disease progression and early detection of HCC.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 201
Pienlainayhtiöiden kulutusluotot vertailussa OPn kulutusluottoihin : Case: OP Etelä-Häme
No full textOpinnäytetyön aiheena oli tutkia ja vertailla OPn vakuudettomia kulutusluottoja pienlainayhtiöiden tarjoamiin kulutusluottoihin. Työssä pyrittiin löytämään vastauksia siihen, miksi asiakkaat monesti ottavat korkeakorkoisia pienlainoja pienlainayhtiöiltä, eivätkä tule hakemaan luottoa pankista. Tutkimusta lähestyttiin teorian kautta kulutusluottojen perustiedoista, lainsäädännöstä ja maksuhäiriöistä sekä haastateltiin toimeksiantajayrityksen työntekijöitä. Teoriaosuuden ja teemahaastatteluiden pohjalta löydettiin vastauksia ja tehtiin johtopäätöksiä työn tutkimusongelmaan. Työ toteutettiin toimeksiantona OP Etelä-Hämeelle.
Työ toteutettiin kahdessa vaiheessa. Tietoperustaa hankittiin kulutusluotoista, pienlainoista, luotonannosta, ihmisten velkaantumisesta, maksuhäiriöistä ja lainsäädännöstä. Teoriapohjan avulla sopivaksi tutkimustavaksi valittiin kvalitatiivinen tutkimusmenetelmä ja laadittiin työhön soveltuvat teemahaastattelukysymykset. Toisena vaiheena toteutettiin haastattelut toimeksiantajayrityksessä. Teemahaastatteluihin valikoitui kolme rahoitusneuvottelijaa OP Etelä-Hämeen toimialueen eri konttoreista. Näin ollen työhön saatiin mielipiteitä ja näkemyksiä rahoitusneuvottelijoilta, joilla jokaisella oli erilainen toimialue ja pankkiura takanaan.
Pienlainayhtiöiden kulutusluottojen helppous nousi suurimmaksi syyksi sille, miksi asiakkaat hakevat lainansa pienlainayhtiöiltä. OPn kulutusluotoissa on kuitenkin huomattavasti matalampi korko, joka voisi toimia etulyöntiasemana kilpailussa. Työstä saatuja tuloksia voidaan hyödyntää toimeksiantajayrityksessä kulutusluotonantoon ja tuomaan vastauksia asiakkaiden tekemiin valintoihin kulutusluottoja hakiessaan.The subject of this thesis was to examine and compare OPs unsecured consumer credits with quickie loan companies’ similar unsecured loans. The aim of this thesis was to find answers why customers so often take high interest rated quickie loans and not come to the bank to get an unsecured consumer credit. The study was approached from the theory of legislation, basic information about unsecured consumer credits and payment defaults and also from interviewing the principal company’s employees. With the theory and the interviews the author was able to find answers and make conclusions to the research problem. The thesis was executed as an assignment for OP Etelä-Häme.
The study was done in two phases. First the author collected background information about consumer credits, quickie loans, lending, peoples getting into debt, payment defaults and legislation. The qualitative research method was chosen for the study and the research questions were set. The empirical phase of this thesis consisted of theme based interviews with three financial negotiators from different branch offices of OP Etelä-Häme. Hence the author gained different kind of opinions and views from different territories and careers in banking.
Quickie loan companies’ easiness was the biggest reason why customers choose rather quickie loans than OPs unsecured consumer credits. However, OPs consumer credits are cheaper than quickie loan companies’ loans. That would be huge advantage in competition. The results from the study can be exploited at OP Etelä-Häme in consumer credits and in answering to the customers’ choices when applying for an unsecured consumer credit
Nucleation kinetics of calcium oxalate monohydrate as a function of pH, magnesium, and osteopontin concentration quantified with droplet microfluidics
No full textA droplet-based microfluidic platform is presented to study the nucleation kinetics of calcium oxalate monohydrate (COM), the most common constituent of kidney stones, while carefully monitoring the pseudo-polymorphic transitions. The precipitation kinetics of COM is studied as a function of supersaturation and pH as well as in the presence of inhibitors of stone formation, magnesium ions (Mg2+), and osteopontin (OPN). We rationalize the trends observed in the measured nucleation rates leveraging a solution chemistry model validated using isothermal solubility measurements. In equimolar calcium and oxalate ion concentrations with different buffer solutions, dramatically slower kinetics is observed at pH 6.0 compared to pHs 3.6 and 8.6. The addition of both Mg2+ and OPN to the solution slows down kinetics appreciably. Interestingly, complete nucleation inhibition is observed at significantly lower OPN, namely, 3.2 × 10-8 M, than Mg2+ concentrations, 0.875 × 10-4 M. The observed inhibition effect of OPN emphasizes the often-overlooked role of macromolecules on COM nucleation due to their low concentration presence in urine. Moreover, analysis of growth rates calculated from observed lag times suggests that inhibition in the presence of Mg2+ cannot be explained solely on altered supersaturation. The presented study highlights the potential of microfluidics in overcoming a major challenge in nephrolithiasis research, the overwhelming physiochemical complexity of urine.Green Open Access added to TU Delft Institutional Repository 'You share, we take care!' - Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.Complex Fluid Processin
Prevention of experimental autoimmune uveoretinitis by blockade of osteopontin with small interfering RNA
No full textOsteopontin (OPN) is elevated during the progression of experimental autoimmune uveoretinitis (EAU) in C57BL/6 (B6) mice. Furthermore, EAU symptoms are ameliorated in OPN knockout mice or in B6 mice treated with anti-OPN antibody (M5). Recently, OPN has been shown to promote the Th1 response not only in the extracellular space as a secretory protein but also in cytosol as a signaling component. Thus, we attempted to reduce OPN in both compartments by using a small interfering RNA (siRNA) targeting the OPN coding sequence (OPN-siRNA). EAU was induced in B6 mice by immunization with human interphotoreceptor retinoid-binding protein (hIRBP) peptide sequence 1-20. The OPN- or control-siRNA was administered with hydrodynamic methods 24 hours before and simultaneously with immunization (prevention regimen). When plasma OPN levels were quantified following siRNA administration with the prevention regimen, the level in the OPN-siRNA-treated group was significantly lower than that in the control-siRNA-treated group. Accordingly, the clinical and histopathological scores of EAU were significantly reduced in B6 mice when siRNA caused OPN blockade. Furthermore, TNF-α, IFN-γ, IL-2, GM-CSF and IL-17 levels in the culture supernatants were markedly suppressed in the OPN-siRNA-treated group, whereas the proliferative responses of T lymphocytes from regional lymph nodes against immunogenic peptides was not significantly reduced. On the other hand, the protection was not significant if the mice received the OPN-siRNA treatment on day 7 and day 8 after immunization when the clinical symptoms appeared overt (reversal regimen). Our results suggest that OPN blockade with OPN-siRNA can be an alternative choice for the usage of anti-OPN antibody and controlling uveoretinitis in the preventive regimen
Recombinant human osteopontin expressed in Nicotiana benthamiana stimulates osteogenesis related genes in human periodontal ligament cells.
No full textTissue engineering aims to utilise biologic mediators to facilitate tissue regeneration. Several recombinant proteins have potential to mediate induction of bone production, however, the high production cost of mammalian cell expression impedes patient access to such treatments. The aim of this study is to produce recombinant human osteopontin (hOPN) in plants for inducing dental bone regeneration. The expression host was Nicotiana benthamiana using a geminiviral vector for transient expression. OPN expression was confirmed by Western blot and ELISA, and OPN was purified using Ni affinity chromatography. Structural analysis indicated that plant-produced hOPN had a structure similar to commercial HEK cell-produced hOPN. Biological function of the plant-produced hOPN was also examined. Human periodontal ligament stem cells were seeded on an OPN-coated surface. The results indicated that cells could grow normally on plant-produced hOPN as compared to commercial HEK cell-produced hOPN determined by MTT assay. Interestingly, increased expression of osteogenic differentiation-related genes, including OSX, DMP1, and Wnt3a, was observed by realtime PCR. These results show the potential of plant-produced OPN to induce osteogenic differentiation of stem cells from periodontal ligament in vitro, and suggest a therapeutic strategy for bone regeneration in the future
The role of osteopontin in tendon tissue remodeling after denervation-induced mechanical stress deprivation
No full textIt has been shown that musculoskeletal tissues undergo dynamic tissue remodeling by a process that is quite sensitive to the mechanical environment. However, the detailed molecular mechanism underlying this process remains unclear. We demonstrate here that after denervation-induced mechanical stress deprivation, tendons undergo dynamic tissue remodeling as evidenced by a significant reduction of the collagen fibril diameter. Importantly, the transient up-regulation of osteopontin (OPN) expression was characteristic during the early phase of tendon tissue remodeling. Following this dynamic change of OPN expression, matrix metalloproteinase (MMP)-13 expression was induced, which presumably accounts for the morphological changes of tendon by degrading tendon collagen fibrils. The modulation of MMP-13 expression by OPN was specific, since the expression of MMP-2, which is also known to be involved in tissue remodeling, did not alter in the tendons under the absence or presence of OPN. We also demonstrate that the modulation of MMP-13 expression by OPN is due to the signaling through cell surface receptors for OPN. Thus, we conclude that OPN plays a crucial role in conveying the effect of denervation-induced mechanical stress deprivation to the tendon fibroblasts to degrade the extracellular matrices by regulating MMP-13 expression in tendon fibroblasts
Osteopontin: role in immune regulation and stress responses
No full textOsteopontin (OPN) is a pluripotent soluble protein found in all body fluids and expressed in many tissues and cells. It stimulates signal transduction pathways via integrins and CD44 variants that regulate various cellular activities including the cell's interaction with extracellular matrix, cell survival and immune responses. OPN deficiency is linked to a reduced Th1 immune response in infectious diseases, autoimmunity and delayed type hypersensitivity. OPN is also involved in various physiological stress situations such as mechanical stress, oxidative stress and cellular stress. However, OPN's role in physical and psychological stress is largely unexplored.
In this dissertation, I used OPN-deficient mice to evaluate OPN's role in physical stress responses and to determine its influence on immune organ lymphocyte homeostasis in response to stress. Using a hindlimb-unloading (HU) stress model, I compared OPN-/- (knockout) mice with OPN+/+ mice subjected to HU for 3 days. Whereas OPN+/+ mice suffered a marked reduction of body weight and significant spleen and thymus atrophy, OPN-/- mice exhibited minor weight loss and much less spleen and thymus atrophy. The HU-induced lymphoid organ atrophy was the result of dramatically diminished numbers of T and B cells in the spleen and CD4+/CD8+ double-positive cells in the thymus of OPN+/+ mice but not in OPN-/- mice. Increased levels of corticosterone, which modulate lymphocyte activation responses and apoptosis during stress, were found only in OPN+/+ mice. Apoptotic cell death was evident in the spleen and thymus tissue of OPN+/+mice subjected to HU but not in OPN-/- mice and untreated controls.
In a different stress model, chronic restraint stress (CRS), I have demonstrated that OPN-deficient mice are resistant to CRS-induced lymphoid organ atrophy and unable to mount a significant up-regulation of corticosterone production in response to stress. Another HPA (Hypothalamus Pituitary Adrenal) axis hormone, ACTH, was also found to be unable to respond to stress challenge in the absence of OPN. Administration of soluble OPN into OPN-/- mice led to a further reduction of lymphoid organ mass in response to stress whereas administration of anti-OPN antibody 2C5 in wild type mice alleviated stress-induced organ atrophy. Taken together, these results indicate that OPN is critical in mediating stress-induced immune organ atrophy, likely through influencing the HPA axis hormone pathway.Ph.D.Includes bibliographical references (p. 102-115)
Prognostic significance of osteopontin expression in gastric cancer: a meta-analysis
No full textBackground: Accumulated studies have exploited the association between osteopontin (OPN) expression and survival of patients with gastric cancer (GC), however, the results were controversial. Thus, we performed a meta-analysis, aiming to investigate the prognostic role of OPN for GC patients and to explore the association between OPN and clinicalpathological features of GC. Results: A total of ten studies involving 1775 patients were included in final meta-analysis. Of the included studies, nine were conducted on Asian patients and one was performed on Caucasian patients. Regarding OPN detection, immunohistochemistry (IHC) was used on tissue specimens in eight studies and enzyme linked immunosorbent assay (ELISA) was used on plasma specimens in two studies. The pooled data showed that high OPN expression was correlated with poor OS (HR = 1.59, 95% CI: 1.15-2.22, p = 0.006). Subgroup analyses demonstrated that OPN had enhanced prognostic value for Asian patients (HR = 1.64, 95% CI = 1.11-2.41, p = 0.012) and for patients receiving surgical resection (HR = 1.6, 95% CI = 1.04-2.48, p = 0.034). In addition, the results also showed that elevated OPN expression was associated with lymph node metastasis, TNM stage, depth of invasion, tumor size and distant metastasis in GC. Methods: Relevant studies were retrieved through PubMed, Embase and Web of Science. Combined hazard ratio (HR) and 95% confidence interval (CI) were calculated to assess the association between OPN and overall survival (OS). Subgroup analyses and publication bias were also conducted. Conclusions: OPN overexpression was correlated with poor OS and clinical features reflecting high aggressiveness in patients with GC. OPN was a promising prognostic biomarker for GC.Clinical Features Research of Capital [Z141107002514160]SCI(E)[email protected]
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