Higher Osteopontin Levels Research Articles

Plasma osteopontin levels in prediction of prognosis in acute myocardial infarction

Objective We sought to explain the clinical importance of the osteopontin (OPN) in the setting of acute ST-elevation myocardial infarction (STEMI).Methods Eighty consecutive patients (55 ± 11 years, 12 women and 68 men) and sixty healthy control subjects were included in the study. In all patients, plasma OPN levels were assessed on admission and on the third day (peak value). Creatinine kinase (CK)/CK-myocardial band (MB), troponin I and N-terminal pro-brain natriuretic factor levels and echocardiographic fi ndings were also recorded. Patients were classifi ed into high and low OPN groups according to the median OPN value, and monitored for the occurrence of major adverse cardiovascular events (MACE).Results Patients with STEMI had higher OPN levels (23.8 [16.7-41.3] ng/ml) on admission than the control subjects (18.0 [11.3-31.5] ng/ml, P= 0.004). The third day value of OPN was signifi cantly higher (39.2 [27.2-56.0] ng/ml) than the OPN level on admission (23.8 [16.7-41.3] ng/ml, P < 0.001). Admission and peak OPN levels were not correlated with CK/CK-MB, white blood cell counts, troponin I and the N-terminal pro-brain natriuretic factor. The plasma OPN levels were not correlated with left ventricular wall motion score index either. In the subgroups of infarct localization and reperfusion strategy, plasma OPN levels were similar. When the patients were compared according to the median OPN values, there were no diff erences in the occurrence of MACE between the high and low OPN groups.Conclusion This study suggests, for the fi rst time, that the plasma OPN level increases in the fi rst hours of the acute STEMI; however, it could not be used as a prognostic biomarker of STEMI.

Plasma and tissue osteopontin in relation to plasma selenium in patients with psoriasis

The association between psoriasis and cardiovascular diseases (CVD) is well documented yet the underlying mechanisms remain unknown. Over-expression of osteopontin (OPN) was reported in plasma of patients with psoriasis; with increased cardiovascular risk factors in these patients. Selenium (Se) compounds are effective in down-regulation of OPN expression. We investigated the levels of OPN and Se in psoriasis, and their relation to metabolic status in patients to identify a possible link between these markers and co-morbidities observed. Plasma and tissue samples from 20 patients with psoriasis and 10 control subjects were collected for enzyme-linked immunosorbent assays. The clinical significance of plasma, tissue OPN and plasma Se levels in patients vs. control subjects was analysed in relation to metabolic disorders. Plasma and tissue OPN were significantly higher in patients than in controls (P < 0.001). Plasma Se levels were significantly lower in patients than in controls (P < 0.001). Elevated plasma OPN levels (≥ 51.10 ng/mL) and depressed plasma Se (≤ 5.19 μg/dL) were significantly associated with the occurrence of psoriasis. Plasma OPN negatively correlated with plasma Se in patients (P = 0.003), but not in controls (P = 0.183). High plasma OPN and low plasma Se levels are predictable factors for occurrence of psoriasis. Further studies examining the effects of Se supplementations on the levels of plasma OPN, together with their effects on psoriasis outcome and cardiovascular risk factors in these patients, are needed.

Osteopontin Splice Variants In Pancreatic Cancer Cell Lines And Fine Needle Aspirates Of Pancreatic Tumors

Introduction: Osteopontin (OPN) is a secreted phosphoprotein that confers on cancer cells a migratory phenotype and activates signaling pathways that induce cell survival, proliferation, invasion, and metastasis. High levels of tissue and serum OPN have been reported to correlate with poor survival in pancreatic ductal adenocarcinoma (PDA) patients. Three isoforms of OPN (OPNa, b and c) are known to exist as a result of alternative splicing of the OPN gene. In this study, we examined the expression of OPN isoforms in PDA cell lines and in fine needle aspirates (FNAs) from pancreatic tumors. We also evaluated their individual effect on PDA cell metastatic potential. Methods: FNAs from 24 patients were collected and analyzed for the expression of OPN isoforms by RT-PCR. Data were evaluated and correlated with different clinicopathological parameters. Basal levels of OPN isoforms in seven PDA cell lines were examined by RT-PCR. Transient transfection studies using cDNA plasmids specific to each isoform analyzed the functional impact of each isoform on PDA cell behavior using wound-induced migration, MTT, and transwell infiltration assays. The effect of knocking down OPN by small hairpin (sh) RNA on cell proliferation and migration potential was also evaluated. Results: FNAs were diagnosed as PDA (n= 21) or non-malignant (n=3). OPNa was highly expressed in 20 of 21 PDA FNAs (95%), compared to 1 of 3 (33%) in non-malignant FNA samples. High levels of OPNb were present in 14 of 21 (67%) of PDA samples, while OPNc was present in 5 of 21 (24%). No OPN b or c was detected in the non-malignant FNAs. In PDA cell lines, all three isoforms were present in BxPC-3, Hs766T, and PK9 cells, which express high levels of total OPN. OPNa and OPNb were found in Panc-1, HPAF-1 and AsPC-1 cells, which express moderate levels of total OPN, and only OPNa was found in MiaPaca cells, which express low levels of total OPN. Transwell infiltration and wound-induced migration assays revealed that OPNb induced MiaPaca cell migration, while OPNa and OPNc had no significant effects. By contrast, OPNc significantly (p<0.05) suppressed the migratory activity of AsPC-1 cells although no significant changes were induced by OPNa or b. OPNc overexpression also induced an early, but significant (P<0.05) reduction of cell proliferation compared to controls. Knocking down total OPN by shRNA resulted in significant (p<0.05) reduction in cell proliferation and migration. Conclusion: The findings of the present study show for the first time the consistent presence of OPNa in PDA FNAs and the consistent absence of OPNb and c in non-malignant FNAs, suggesting a discriminatory role for the individual isoforms. Our functional in vitro data demonstrate that OPN splice variants differentially modulate the migratory property of PDA cells and suggest that this may be one of the mechanisms underlying the pathologic heterogeneity of PDA progression.

Osteopontin concentrations are increased in cerebrospinal fluid during attacks of multiple sclerosis

Background:The cytokine osteopontin (OPN) is a potential key player in the immunopathogenesis of multiple sclerosis (MS) and a candidate biomarker for disease activity. Objective:The objective of this study was to examine concentrations of OPN in the cerebrospinal fluid (CSF) across the clinical spectrum of MS. Methods:Our research consisted of a cross-sectional study of patients from two randomized, placebo-controlled trials. Concentrations of OPN and other blood and CSF markers were determined using an enzyme-linked immunosorbent assay (ELISA). Samples were obtained from untreated patients with exacerbation of clinically isolated syndrome (CIS) (n = 25) and relapsing–remitting MS (RRMS) (n = 41) of whom 48 participated in clinical trials, randomly allocated to treatment with placebo or methylprednisolone (MP) and undergoing repeated sampling after 3 weeks. Furthermore, we obtained CSF and blood samples from patients with primary progressive MS (PPMS, n = 9), secondary progressive MS (SPMS, n = 28) and other neurological disorders (OND, n = 44), and blood samples from 24 healthy subjects. Results:OPN concentrations were significantly increased in the CSF of patients with CIS (p = 0.02) and RRMS (p < 0.001) in exacerbation compared to patients with OND, and increased levels of OPN were associated with high values of other biomarkers of inflammation. At 3-week follow-up CSF OPN concentrations had decreased significantly from baseline regardless treatment with placebo or MP. Patients with PPMS had increased OPN levels in the CSF (p = 0.004) and high CSF levels of OPN were associated with high degrees of disability. Conclusions:OPN concentration in the CSF is a dynamic indicator of disease activity in RRMS, presumably reflecting ongoing inflammation. Increased CSF OPN concentrations in PPMS may indicate ongoing inflammation even in these patients.

Effects of osteopontin inhibition on radiosensitivityof MDA-MB-231 breast cancer cells

BackgroundOsteopontin (OPN) is a secreted glycophosphoprotein that is overexpressed in various tumors, and high levels of OPN have been associated with poor prognosis of cancer patients. In patients with head and neck cancer, high OPN plasma levels have been associated with poor prognosis following radiotherapy. Since little is known about the relationship between OPN expression and radiosensitivity, we investigated the cellular and radiation induced effects of OPN siRNA in human MDA-MB-231 breast cancer cells.MethodsMDA-MB-231 cells were transfected with OPN-specific siRNAs and irradiated after 24 h. To verify the OPN knockdown, we measured the OPN mRNA and protein levels using qRT-PCR and Western blot analysis. Furthermore, the functional effects of OPN siRNAs were studied by assays to assess clonogenic survival, migration and induction of apoptosis.ResultsTreatment of MDA-MB-231 cells with OPN siRNAs resulted in an 80% decrease in the OPN mRNA level and in a decrease in extracellular OPN protein level. Transfection reduced clonogenic survival to 42% (p = 0.008), decreased the migration rate to 60% (p = 0.15) and increased apoptosis from 0.3% to 1.7% (p = 0.04). Combination of OPN siRNA and irradiation at 2 Gy resulted in a further reduction of clonogenic survival to 27% (p < 0.001), decreased the migration rate to 40% (p = 0.03) and increased apoptosis to 4% (p < 0.005). Furthermore, OPN knockdown caused a weak radiosensitization with an enhancement factor of 1.5 at 6 Gy (p = 0.09) and a dose modifying factor (DMF10) of 1.1.ConclusionOur results suggest that an OPN knockdown improves radiobiological effects in MDA-MB-231 cells. Therefore, OPN seems to be an attractive target to improve the effectiveness of radiotherapy.

Abstract A16: Thrombin promotes hepatocellular carcinoma metastasis via mediating osteopontin-dependent tumor growth and adhesion activity

Abstract In our previous study, osteopontin (OPN) was identified as one of the leading genes that promote the metastasis of hepatocellular carcinoma (HCC), and a potential therapeutic target for HCC metastasis. The serine protease thrombin can interact with OPN and modify its biologic activity. To explore the role of thrombin alone or collaborating with OPN in HCC, we investigated the thrombin level and its correlation to the prognosis of 72 HCCs with different OPN backgrounds using real-time PCR and Western blot, and evaluated the effects of OPN fragments formed by thrombin cleavage on in vitro proliferation and adhesion of HCC cells. We found that the expression level of thrombin was positively related to the metastatic potentials of HCC cell lines, and was remarkably overexpressed in HCCs compared with the adjacent nontumor tissues. In addition, HCC tissues from patients with recurrent disease displayed much higher thrombin levels, particularly in those with OPN overexpressed. In the immunohistochemical study of 230 HCCs, only in those with a higher OPN level, a significant association was found between the higher thrombin level and the overall survival (OS; P &amp;lt; 0.01) and time to recurrence (TTR; P &amp;lt; 0.0001) of patients. Assays in vitro demonstrated that thrombin could promote the proliferation and adhesion of OPN+ HCC cells. And N-terminal of OPN showed better effects on the proliferation and adhesion abilities of HCC cells than the intact protein. Thrombin treatment could activate FAK pathway of OPN+ HCC cells, and this activation was blocked by the inhibition of integrin β1. Conclusions: Thrombin plays an important role in OPN-mediated aggressive phenotype and poor prognosis of HCC, which may be through the activation of the integrin β1 -FAK signaling. So, thrombin might be a potential therapeutic target to combat HCC progression in thrombin+/OPN+ patients. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A16.

Osteopontin Expressed in Tubular Epithelial Cells Regulates NK Cell-Mediated Kidney Ischemia Reperfusion Injury

Renal ischemia reperfusion injury (IRI) occurs after reduced renal blood flow and is a major cause of acute injury in both native and transplanted kidneys. Studies have shown diverse cell types in both the innate and the adaptive immune systems participate in kidney IRI as dendritic cells, macrophages, neutrophils, B cells, CD4(+) NK(+) cells, and CD4(+) T cells all contribute to this form of injury. Recently, we have found that NK cells induce apoptosis in tubular epithelial cells (TECs) and also contribute to renal IRI. However, the mechanism of NK cell migration and activation during kidney IRI remains unknown. In this study, we have identified that kidney TECs express a high level of osteopontin (OPN) in vitro and in vivo. C57BL/6 OPN-deficient mice have reduced NK cell infiltration with less tissue damage compared with wild-type C57BL/6 mice after ischemia. OPN can directly activate NK cells to mediate TEC apoptotic death and can also regulate chemotaxis of NK cells to TECs. Taken together, our study's results indicate that OPN expression by TECs is an important factor in initial inflammatory responses that involves NK cells activity in kidney IRI. Inhibiting OPN expression at an early stage of IRI may be protective and preserve kidney function after transplantation.

Role of tissue inhibitor of metalloproteinases-1 in the development of autoimmune lymphoproliferation

Inherited defects decreasing function of the Fas death receptor cause autoimmune lymphoproliferative syndrome and its variant Dianzani's autoimmune lymphoproliferative disease. Analysis of the lymphocyte transcriptome from a patient with this latter condition detected striking over-expression of osteopontin and tissue inhibitor of metalloproteinases-1. Since previous work on osteopontin had detected increased serum levels in these patients, associated with variations of its gene, the aim of this work was to extend the analysis to tissue inhibitor of metalloproteinases-1. Tissue inhibitor of metalloproteinases-1 levels were evaluated in sera and culture supernatants from patients and controls by enzyme-linked immunosorbent assay. Activation- and Fas-induced cell death were induced, in vitro, using anti-CD3 and anti-Fas antibodies, respectively. Tissue inhibitor of metalloproteinases-1 levels were higher in sera from 32 patients (11 with autoimmune lymphoproliferative syndrome and 21 with Dianzani's autoimmune lymphoproliferative disease) than in 50 healthy controls (P<0.0001), unassociated with variations of the tissue inhibitor of metalloproteinases-1 gene. Both groups of patients also had increased serum levels of osteopontin. In vitro experiments showed that osteopontin increased tissue inhibitor of metalloproteinases-1 secretion by peripheral blood monocytes. Moreover, tissue inhibitor of metalloproteinases-1 significantly inhibited both Fas- and activation-induced cell death of lymphocytes. These data suggest that high osteopontin levels may support high tissue inhibitor of metalloproteinases-1 levels in autoimmune lymphoproliferative syndrome and Dianzani's autoimmune lymphoproliferative disease, and hence worsen the apoptotic defect in these diseases.

Identification of Potential Serum Biomarkers of Glioblastoma: Serum Osteopontin Levels Correlate with Poor Prognosis

The aim of this study is to identify serum biomarkers with classification and prognosis utility for astrocytoma, in particular glioblastoma (GBM). Our previous glioma microarray database was mined to identify genes that encode secreted or membrane-localized proteins. Subsequent analysis was done using significant analysis of microarrays, followed by reverse transcription-quantitative PCR (RT-qPCR) and immunohistochemical validation in tumor tissues, ELISA and Western blot validation in sera, and correlation with survival of GBM patients. Significant analysis of microarrays identified 31 upregulated and 3 downregulated genes specifically in GBMs. RT-qPCR validation on an independent set of samples confirmed the GBM-specific differential expression of several genes, including three upregulated (CALU, CXCL9, and TIMP1) and two downregulated (GPX3 and TIMP3) novel genes. With respect to osteopontin (OPN), we show the GBM-specific upregulation by RT-qPCR and immunohistochemical staining of tumor tissues. Elevated serum OPN levels in GBM patients were also shown by ELISA and Western blot. GBM patients with high serum OPN levels had poorer survival than those with low serum OPN levels (median survival 9 versus 22 months respectively; P = 0.0001). Further, we also show high serum TIMP1 levels in GBM patients compared with grade II/III patients by ELISA and downregulation of serum GPX3 and TIMP3 proteins in GBMs compared with normal control by Western blot analysis. Several novel potential serum biomarkers of GBM are identified and validated. High serum OPN level is found as a poor prognostic indicator in GBMs. Identified serum biomarkers may have potential utility in astrocytoma classification and GBM prognosis.

Expression and prognostic significance of osteopontin and caspase‐3 in hepatocellular carcinoma patients after curative resection

Osteopontin (OPN) plays an important role in the development, invasion, and metastasis of malignancies. Recently, several studies have reported that OPN enhances chemoresistance in small-cell lung cancer and breast cancer by blocking caspase-9 and caspase-3-dependent cell apoptosis. The aim of this study was to assess the value of OPN and caspase-3 for predicting tumor recurrence after curative resection in hepatocellular carcinoma (HCC) patients. We found that OPN expression increased concordantly with increasing metastatic potential in human HCC cell lines, whereas caspase-3 expression declined. In a tumor tissue microarray immunohistochemical analysis, we found that patients with higher levels of OPN and lower levels of caspase-3 had a significantly poorer prognosis than patients with lower OPN and higher caspase-3 levels. The combination of OPN and caspase-3 expression thus served as an effective prognosticator. These findings suggest that OPN alone or in combination with caspase-3 may act as an independent indicator for HCC patients after curative resection.

Increased serum osteopontin is a risk factor for osteoporosis in menopausal women

Osteopontin (OPN)-deficient mice are resistant to ovariectomy-induced osteoporosis. Therefore, we hypothesized that women with OPN overexpression may show less resistance to postmenopausal osteoporosis. In this study, we first demonstrated that serum OPN levels could be used as a biomarker for the early diagnosis of osteoporosis in postmenopausal women. Animal studies indicate that OPN-deficient mice are resistant to ovariectomy-induced osteoporosis. From 2004 to 2006, 124 women over the age of 45 were enrolled in a menopausal group, while another 95 women, from 25 to 45 years of age with regular menstruation, were enrolled into a childbearing age group. The serum concentrations of OPN were calculated using the enzyme-link immunosorbent assay method, and bone mineral densities were determined with dual energy X-ray absorptiometry. Serum OPN levels had a significant positive correlation with age (menopausal group, p < 0.0001) and a negative correlation with body weight, height, hip bone mineral density, and T-scores in the menopausal group. In contrast, there was a positive correlation with the E2 concentration and height, but there was no significant association with the above variables in the childbearing age group. Additionally, high serum OPN levels (>14.7 ng/ml) was a significant risk factor causing menopausal osteoporosis (odds ratio = 2.96, 95% confidence interval, 1.055-8.345). Serum OPN levels could be used as a biomarker for the early diagnosis of osteoporosis in postmenopausal women.

Osteopontin: An Effector and an Effect of Tumor Metastasis

Osteopontin (OPN) is a matricellular protein that is produced by multiple tissues in our body and is most abundant in bone. It is also produced by cancer cells and plays a determinative role in the growth, progression and metastasis of cancer. Clinically, OPN has been reported to be upregulated in tumor cells per se; this is also reflected by increased levels of OPN in the circulation. Thus, increased OPN levels the plasma are an effect of tumor growth and progression. Functionally, high OPN levels are determinative of higher incidence of bone metastases in mouse models and are clinically correlated with metastatic bone disease and bone resorption in advanced breast cancer patients. Several research efforts have been made to therapeutically target and inhibit the activities of OPN. In this article we have reviewed OPN in its role as an effector of critical steps in tumor progression and metastasis, with a particular emphasis on its role in facilitating bone metastasis of breast cancer. We have also addressed the role of the host-derived OPN in influencing the malignant behavior of the tumor cells.

Associations between plasma osteopontin levels and the severities of coronary and aortic atherosclerosis

High levels of osteopontin (OPN) mRNA and proteins were reported in atherosclerotic plaques [1–3]. OPN-overexpressing transgenic mice developed marked atherosclerotic lesions [4]. These suggest that OPN plays an important role in atherosclerosis. Regarding blood OPN levels, we reported plasma OPN levels to be higher in patients with coronary artery disease (CAD) than without CAD [5]. However, some overlap in OPN levels was found between patients with and without CAD. Since atherosclerotic process is a generalized process that may involve the entire vasculature, we hypothesized that plasma OPN levels may reflect not only coronary atherosclerosis but also atherosclerosis in other vascular beds.

Antigen-Specific Induction of Osteopontin Contributes to the Chronification of Allergic Contact Dermatitis

Allergic contact dermatitis is a T cell-mediated immune response, which in its relapsing chronic form is of high socioeconomic impact. The phosphoglycoprotein osteopontin (OPN) has chemotactic and Th1 cytokine functions and in various models is essential for robust T cell-mediated immunity. Here we demonstrate that OPN is abundantly expressed by both effector T cells and keratinocytes in allergic contact dermatitis lesions. T cells from nickel-allergic donors secrete high levels of OPN following antigen-specific stimulation. OPN may substitute for missing IFN-gamma secretion in T effector cells because low IFN-gamma-producing T cell clones secrete high levels of OPN, and OPN down-modulates their interleukin-4 expression. Furthermore, interferon-gamma from T effector cells augments OPN in allergic contact dermatitis by inducing OPN in keratinocytes, which in turn polarizes dendritic cells and attracts inflammatory cells. In the murine contact hypersensitivity (CHS) model for allergic contact dermatitis, OPN is strongly induced in antigen-specific proliferating T cells, and OPN null mice display a reduced chronic CHS inflammatory response due to a decreased influx of effector T cells. Importantly, because of its function for chronic allergic contact dermatitis, OPN may well be a therapeutic target, because anti-OPN antibody treatment in part suppresses established chronic CHS.

Increased plasma levels of osteopontin are associated with activation of the renin–aldosterone system and with myocardial and coronary microvascular damage in dilated cardiomyopathy

In patients with dilated cardiomyopathy (DCM) abnormal myocardial blood flow (MBF) has been associated with coronary microvascular dysfunction. The aim of this study was to test the hypothesis that osteopontin (OPN) plasma levels could be associated with the activation of the renin–aldosterone system (RAS) in these patients and be involved in mediating myocardial and coronary damage. In 66 patients with idiopathic left ventricular dysfunction of variable severity the plasma levels of OPN were correlated with biomarkers of systemic metabolism, RAS activation, myocardial dysfunction and with clinical indexes of left ventricle (LV) function and perfusion obtained by 2D-echocardiography and PET. As compared to controls, patients showed a significant increase of inflammatory markers (OPN: 508 ± 30.8 ng/ml vs. 426.9 ± 16.4, p < 0.05 and interleukin (IL)-6: 1.71 ± 0.29 pg/ml vs. 0.38 ± 0.03 pg/ml, p < 0.001) and of indexes of cardiac damage. OPN levels were significantly correlated with the extent of microvascular dysfunction (MBF at rest: p = 0.01; during dipyridamole: p = 0.0003) and with plasma renin activity (PRA) ( r = 0.26, p = 0.04). Both in patients with milder or more severe LV dysfunction lower MBF values were associated with higher OPN levels and PRA. These results suggest a interdependent role of RAS and vascular inflammation in cardiomyopathy.

Considerable variation in the concentration of osteopontin in human milk, bovine milk, and infant formulas

Osteopontin (OPN) is a multifunctional bioactive protein that is implicated in numerous biological processes such as bone remodeling, inhibition of ectopic calcification, and cellular adhesion and migration, as well as several immune functions. Osteopontin has cytokine-like properties and is a key factor in the initiation of T helper 1 immune responses. Osteopontin is present in most tissues and body fluids, with the highest concentrations being found in milk. In the present study, ELISA for human and bovine milk OPN were developed and OPN concentration in human breast milk, bovine milk, and infant formulas was measured and compared. The OPN concentration in human milk was measured to approximately 138mg/L, which corresponds to 2.1% (wt/wt) of the total protein in human breast milk. This is considerably higher than the corresponding OPN concentrations in bovine milk (∼18mg/L) and infant formulas (∼9mg/L). Moreover, bovine milk OPN is shown to induce the expression of the T helper 1 cytokine IL-12 in cultured human lamina propria mononuclear cells isolated from intestinal biopsies. Finally, the OPN concentration in plasma samples from umbilical cords, 3-mo-old infants, and pregnant and nonpregnant adults was measured. The OPN level in plasma from 3-mo-old infants and umbilical cords was found to be 7 to 10 times higher than in adults. Thus, the high levels of OPN in milk and infant plasma suggest that OPN is important to infants and that ingested milk OPN is likely to induce cytokine production in neonate intestinal immune cells.

Plasma Osteopontin Levels in Patients With Head and Neck Cancer Undergoing Chemoradiotherapy

To explore the prognostic role of plasma levels of osteopontin (OPN), a phosphoglycoprotein with adhesive properties, in patients with head and neck squamous cell carcinoma (HNSCC) undergoing concomitant chemoradiotherapy. Previous studies have proposed OPN level as a prognostic factor in several cancers. Prospective analysis of plasma OPN levels, before and within 12 weeks after treatment, in a cohort of patients with HNSCC undergoing platinum-based chemoradiotherapy at our center. Academic center. Sixty-nine patients diagnosed as having HNSCC. Plasma levels of OPN were assessed before the start and after the conclusion of chemoradiotherapy by using an enzyme-linked immunosorbency assay kit. Chemoradiotherapy was exclusive (n = 52) or adjuvant to surgery (n = 17). Levels of OPN were correlated with clinicopathological characteristics, response to treatment, and overall survival. Pretreatment plasma OPN levels were higher in patients with advanced T and N stages compared with patients with early stages (P = .009 and .07, respectively). Mean (SD) plasma levels of OPN measured before (102.5 [68.1] ng/mL) and after (104.0 [53.6] ng/mL) treatment did not differ (P = .18, paired t test). Pretreatment and posttreatment levels of OPN were lower in patients who achieved a complete response compared with those who failed to respond (75.0 [41.5] vs 131.2 [82.9] ng/mL [P = .005] and 86.8 [40.5] vs 141.6 [58.4] ng/mL [P = .004], respectively). Patients with high pretreatment OPN levels (>82.1 ng/mL) had shorter survival time (P < .001). Posttreatment OPN levels were marginally (P = .10) associated with survival time in univariate analysis. In patients with HNSCC undergoing chemoradiotherapy, a low pretreatment plasma OPN level is associated with treatment response and better survival. Modulation of OPN levels by chemoradiotherapy may also be associated with outcome. Further studies with serial measurement of OPN levels are warranted in these patients.

Osteopontin promotes fibrosis in dystrophic mouse muscle by modulating immune cell subsets and intramuscular TGF-β

Duchenne muscular dystrophy (DMD) is an X-linked, degenerative muscle disease that is exacerbated by secondary inflammation. Here, we characterized the immunological milieu of dystrophic muscle in mdx mice, a model of DMD, to identify potential therapeutic targets. We identified a specific subpopulation of cells expressing the Vbeta8.1/8.2 TCR that is predominant among TCR-beta+ T cells. These cells expressed high levels of osteopontin (OPN), a cytokine that promotes immune cell migration and survival. Elevated OPN levels correlated with the dystrophic process, since OPN was substantially elevated in the serum of mdx mice and muscle biopsies after disease onset. Muscle biopsies from individuals with DMD also had elevated OPN levels. To test the role of OPN in mdx muscle, mice lacking both OPN and dystrophin were generated and termed double-mutant mice (DMM mice). Reduced infiltration of NKT-like cells and neutrophils was observed in the muscle of DMM mice, supporting an immunomodulatory role for OPN in mdx muscle. Concomitantly, an increase in CD4+ and FoxP3+ Tregs was also observed in DMM muscle, which also showed reduced levels of TGF-beta, a known fibrosis mediator. These inflammatory changes correlated with increased strength and reduced diaphragm and cardiac fibrosis. These studies suggest that OPN may be a promising therapeutic target for reducing inflammation and fibrosis in individuals with DMD.

Nitric oxide participation in granulomatous response induced by Paracoccidioides brasiliensis infection in mice

The role of nitric oxide (NO) in granulomas of Paracoccidioides brasiliensis-infected inducible NO synthase-deficient C57BL/6 mice (iNOS KO) and their wild-type counterparts and its association with osteopontin (OPN) and matrix metalloproteinases (MMPs) was studied. At 15 days after infection (DAI), iNOS KO mice showed compact and necrotic granulomas with OPN+ macrophages and multinucleated giant cells, whereas wild-type mice developed loose granulomas with many fungi and OPN+ cells distributed throughout the tissue. In addition, high OPN levels and fungal load were observed in iNOS KO mice. Both experimental groups had MMP-9 activity. At 120 DAI, iNOS KO had smaller granulomas with OPN+ cells, lower OPN levels, lower fungal load and decreased MMP-9 activity compared with wild-type mice. These findings suggest that NO has an important role in granuloma modulation, by controlling OPN and MMP production, as well as by inducing loose granulomas formation and fungal dissemination, resulting, at later phases, in progression of paracoccidioidomycosis.

OPN expression by epithelial tubular cells regulates NK cell-mediated kidney ischemia reperfusion injury (98.30)

Abstract Renal ischemia reperfusion injury (IRI) is a major cause of acute injury in both native and transplanted kidneys. This type of kidney injury is considered an antigen-independent inflammatory condition that involves multiple factors leading to tubular and endothelial dysfunction. We have recently found that NK cells induce apoptosis in tubular epithelial cells (TEC) and contribute to renal IRI. Kidney can express osteopontin (OPN) during injury. Therefore we examined the role OPN in NK cell function and kidney IRI. We have found that TEC expressed high levels of OPN in vitro and in vivo after injury. Kidneys in OPN-/- mice had less severity of IRI compared to wild-type mice (P&amp;lt;0.05). Interestingly, recombinant OPN could activate NK cells that express high levels of perforin and granzyme and could mediate TEC apoptotic death. Importantly, we have found that NK cell migrate towards OPN protein as well as OPN-producing TEC in the transwell assay. Whereas, less NK cell migration was seen towards OPN-/- TEC (P&amp;lt;0.01). Further more, Kidneys in OPN-/- mice had less NK cell infiltration after IRI compared to WT mice (P&amp;lt;0.02). Taken together, our study results support a previously unrecognized role for TEC expression of OPN in NK cell-mediated kidney injury. TEC expression of OPN promotes early kidney inflammatory and IRI, and limiting OPN expression may improve kidney function and graft survival.