Identifying the presence of magnetite in an ensemble of iron-oxide nanoparticles: a comparative neutron diffraction study between bulk and nanoscale.

Scientific interest in iron-oxides and in particular magnetite has been renewed due to the broad scope of their fascinating properties, which are finding applications in electronics and biomedicine. Specifically, iron oxide nanoparticles (IONPs) are gathering attraction in biomedicine. Their cores are usually constituted by a mixture of maghemite and magnetite phases. In view of this, to fine-tune the properties of an ensemble of IONPs towards their applications, it is essential to enhance mass fabrication processes towards the production of monodisperse IONPs with controlled size, shape, and stoichiometry. We exploit the vacancy sensitivity of the Verwey transition to detect the presence of magnetite. Here we provide direct evidence for the Verwey transition in an ensemble of IONPs through neutron diffraction. This transition is observed as a variation in the Fe magnetic moment at octahedral sites and, in turn, gives rise to a change of the net magnetic moment. Finally, we show this variation as the microscopic ingredient driving the characteristic kink that hallmarks the Verwey transition in thermal variation of magnetization.

Targeted delivery of antisense oligonucleotides to pancreatic ß-cells.

Antisense oligonucleotide (ASO) silencing of the expression of disease-associated genes is an attractive novel therapeutic approach, but treatments are limited by the ability to deliver ASOs to cells and tissues. Following systemic administration, ASOs preferentially accumulate in liver and kidney. Among the cell types refractory to ASO uptake is the pancreatic insulin-secreting ß-cell. Here, we show that conjugation of ASOs to a ligand of the glucagon-like peptide-1 receptor (GLP1R) can productively deliver ASO cargo to pancreatic ß-cells both in vitro and in vivo. Ligand-conjugated ASOs silenced target genes in pancreatic islets at doses that did not affect target gene expression in liver or other tissues, indicating enhanced tissue and cell type specificity. This finding has potential to broaden the use of ASO technology, opening up novel therapeutic opportunities, and presents an innovative approach for targeted delivery of ASOs to additional cell types.

Influence of clustering on the magnetic properties and hyperthermia performance of iron oxide nanoparticles.

Clustering of magnetic nanoparticles can drastically change their collective magnetic properties, which in turn may influence their performance in technological or biomedical applications. Here, we investigate a commercial colloidal dispersion (FeraSpinTMR), which contains dense clusters of iron oxide cores (mean size around 9 nm according to neutron diffraction) with varying cluster size (about 18-56 nm according to small angle x-ray diffraction), and its individual size fractions (FeraSpinTMXS, S, M, L, XL, XXL). The magnetic properties of the colloids were characterized by isothermal magnetization, as well as frequency-dependent optomagnetic and AC susceptibility measurements. From these measurements we derive the underlying moment and relaxation frequency distributions, respectively. Analysis of the distributions shows that the clustering of the initially superparamagnetic cores leads to remanent magnetic moments within the large clusters. At frequencies below 105 rad s-1, the relaxation of the clusters is dominated by Brownian (rotation) relaxation. At higher frequencies, where Brownian relaxation is inhibited due to viscous friction, the clusters still show an appreciable magnetic relaxation due to internal moment relaxation within the clusters. As a result of the internal moment relaxation, the colloids with the large clusters (FS-L, XL, XXL) excel in magnetic hyperthermia experiments.

Synthesis methods to prepare single- and multi-core iron oxide nanoparticles for biomedical applications.

We review current synthetic routes to magnetic iron oxide nanoparticles for biomedical applications. We classify the different approaches used depending on their ability to generate magnetic particles that are either single-core (containing only one magnetic core, i.e. a single domain nanocrystal) or multi-core (containing several magnetic cores, i.e. single domain nanocrystals). The synthesis of single-core magnetic nanoparticles requires the use of surfactants during the particle generation, and careful control of the particle coating to prevent aggregation. Special attention has to be paid to avoid the presence of any toxic reagents after the synthesis if biomedical applications are intended. Several approaches exist to obtain multi-core particles based on the coating of particle aggregates; nevertheless, the production of multi-core particles with good control of the number of magnetic cores per particle, and of the degree of polydispersity of the core sizes, is still a difficult task. The control of the structure of the particles is of great relevance for biomedical applications as it has a major influence on the magnetic properties of the materials.

Proteomics analysis of melanoma metastases: association between S100A13 expression and chemotherapy resistance.

BACKGROUND: Disseminated cutaneous malignant melanoma (CMM) is commonly unresponsive to standard chemotherapies, and there are as yet no predictive markers of therapy response. METHODS: In the present study we collected fresh-frozen pretreatment lymph-node metastasis samples (n=14) from melanoma patients with differential response to dacarbazine (DTIC) or temozolomide (TMZ) chemotherapy, to identify proteins with an impact on treatment response. We performed quantitative protein profiling using tandem mass spectrometry and compared the proteome differences between responders (R) and non-responders (NR), matched for age, gender and histopathological type of CMM. RESULTS: Biological pathway analyses showed several signalling pathways differing between R vs NR, including Rho signalling. Gene expression profiling data was available for a subset of the samples, and the results were compared with the proteomics data. Four proteins with differential expression between R and NR were selected for technical validation by immunoblotting (ISYNA1, F13A1, CSTB and S100A13), and CSTB and S100A13 were further validated on a larger sample set by immunohistochemistry (n=48). The calcium binding protein S100A13 was found to be significantly overexpressed in NR compared with R in all analyses performed. CONCLUSIONS: Our results suggest that S100A13 is involved in CMM resistance to DTIC/TMZ.

Surgical nurses' attitudes towards caring for patients dying of cancer - a pilot study of an educational intervention on existential issues.

This is a randomised controlled pilot study using a mixed methods design. The overall aim was to test an educational intervention on existential issues and to describe surgical nurses' perceived attitudes towards caring for patients dying of cancer. Specific aims were to examine whether the educational intervention consisting of lectures and reflective discussions, affects nurses' perceived confidence in communication and to explore nurses' experiences and reflections on existential issues after participating in the intervention. Forty-two nurses from three surgical wards at one hospital were randomly assigned to an intervention or control group. Nurses in both groups completed a questionnaire at equivalent time intervals: at baseline before the educational intervention, directly after the intervention, and 3 and 6 months later. Eleven face-to-face interviews were conducted with nurses directly after the intervention and 6 months later. Significant short-term and long-term changes were reported. Main results concerned the significant long-term effects regarding nurses' increased confidence and decreased powerlessness in communication, and their increased feelings of value when caring for a dying patient. In addition, nurses described enhanced awareness and increased reflection. Results indicate that an understanding of the patient's situation, derived from enhanced awareness and increased reflection, precedes changes in attitudes towards communication.

Comparative expression profiling of distinct T cell subsets undergoing oxidative stress.

The clinical outcome of adoptive T cell transfer-based immunotherapies is often limited due to different escape mechanisms established by tumors in order to evade the hosts' immune system. The establishment of an immunosuppressive micromilieu by tumor cells along with distinct subsets of tumor-infiltrating lymphocytes is often associated with oxidative stress that can affect antigen-specific memory/effector cytotoxic T cells thereby substantially reducing their frequency and functional activation. Therefore, protection of tumor-reactive cytotoxic T lymphocytes from oxidative stress may enhance the anti-tumor-directed immune response. In order to better define the key pathways/proteins involved in the response to oxidative stress a comparative 2-DE-based proteome analysis of naïve CD45RA(+) and their memory/effector CD45RO(+) T cell counterparts in the presence and absence of low dose hydrogen peroxide (H(2)O(2)) was performed in this pilot study. Based on the profiling data of these T cell subpopulations under the various conditions, a series of differentially expressed spots were defined, members thereof identified by mass spectrometry and subsequently classified according to their cellular function and localization. Representative targets responding to oxidative stress including proteins involved in signaling pathways, in regulating the cellular redox status as well as in shaping/maintaining the structural cell integrity were independently verified at the transcript and protein level under the same conditions in both T cell subsets. In conclusion the resulting profiling data describe complex, oxidative stress-induced, but not strictly concordant changes within the respective expression profiles of CD45RA(+) and CD45RO(+) T cells. Some of the differentially expressed genes/proteins might be further exploited as potential targets toward modulating the redox capacity of the distinct lymphocyte subsets thereby providing the basis for further studies aiming at rendering them more resistant to tumor micromilieu-induced oxidative stress.

High expression of GCLC is associated with malignant melanoma of low oxidative phenotype and predicts a better prognosis.

Reactive oxygen species (ROS) are strongly implicated in melanoma development, and treatment with antioxidants has shown efficacy in suppressing malignant transition and progression. Here, we investigated the significance of the glutamate-L: -cysteine ligase catalytic subunit (GCLC) expression, a key regulator of glutathione synthesis, for malignant melanoma. A large set of melanoma cell lines (n = 36) was analyzed, and higher GCLC levels were associated with lower presence of intracellular ROS and interestingly also lower rates of cell proliferation. Moreover, treatment with the antioxidant N-acetylcysteine efficiently reduced the growth speed of several investigated malignant cells. In addition GCLC expression was significantly linked to a prominent set of cellular antioxidants, accounting for the observed lower basal levels of oxidative stress and higher antioxidative capacity. Key attributes defining the malignant phenotype of melanoma cells including survival, invasiveness, and switch from E-cadherin to N-cadherin expression were more prominent in cells with lower GCLC expression. Our findings were further corroborated by observations in Rag2(-/-)γc(-/-)mice, in which melanoma cells with lower GCLC expression depicted a dramatically stronger tumor growth. Furthermore, prognostic significance of GCLC expression was investigated in patients (n = 28) with advanced malignant melanoma. High tumor immunoreactivity for GCLC was a significant determinant for better 5-year overall survival. Conclusively, we show for the first time that GCLC may serve a dual role, as a surrogate marker for cellular redox state as well as malignant potential of melanoma cells. These promising results regarding its prognostic significance as well as its potential as a pharmacological target require further in-depth investigations.

Nuclear HER3 is associated with favorable overall survival in uveal melanoma.

HER3 is a member of the epidermal growth factor receptor (EGFR) family and is expressed in several types of cancer. Both the cytoplasmic and nuclear appearances of the receptor have been reported. Here, we investigate the expression and subcellular distribution of HER3 in uveal melanoma (UM) cells and tissues and its potential impact on clinical outcome of patients. Paraffin-embedded samples from 128 consecutive UM patients, enucleated without alternative treatment on UM diagnosis, were evaluated for HER3 using immunohistochemistry. Immunoreactivity was scored for frequency, intensity of positive cells, and subcellular distribution. The results were correlated with the established clinicopathological parameters using univariate and multivariate statistical analyses. HER3 expression was shown in 70% of the cases (89/128). This contrasts with the other EGFR family receptors (EGFR, HER2 and HER4) that are infrequently expressed in UM. Surprisingly, HER3 was found to be localized solely in the cell nuclei in 56 cases. The remaining 33 HER3 positive cases showed diffuse distribution (cytoplasmic ± nuclear). Nuclear HER3 was independently correlated with a more favorable overall survival (p = 0.043 and hazard ratio = 0.618) compared to cases with diffuse and/or no HER3. Nuclear localization of HER3 was also confirmed in fresh UM material and in UM cell lines. In conclusion, HER3 is frequently localized solely in the cell nuclei in UM and as such it predicts a more favorable overall survival.

Piglet model for studying esophageal regrowth after resection and interposition of a silicone stented small intestinal submucosa tube.

PURPOSE: To investigate the use and subsequent healing of a silicone stented small intestinal submucosa (SIS) tube as a full-circumference replacement following surgical resection of the esophagus in piglets. MATERIAL AND METHODS: Three centimeters of the intrathoracic esophagus was replaced with a silicone stented SIS tube (Cook Medical) in 6 growing piglets. The esophageal stent was retained for 4 weeks. Esophageal dilations were performed, if needed, after stent removal. RESULTS: The piglets were sacrificed 1-17 weeks after surgery. Recurrent dilations were needed after stent removal. Histology showed that the gap between the resection margins was filled with new loose connective tissue consisting of fibroblasts and few inflammatory cells. In this tissue, intense angiogenesis was seen at the early time points, which then gave way to the proliferation of immature-looking smooth-muscle-like cells in the submucosa, which appeared to stem from the pericytes of the ingrowing capillaries. CONCLUSIONS: Through using a stented SIS tube as a circumferential esophageal replacement in a piglet model, this study suggests that pericytes from ingrowing capillaries may play a role in the remodeling of the SIS mesh. It remains to be seen if this process gives a favorable end result because stricture formation after stent removal remains a problem.

Multipotent mesenchymal stromal cells express FoxP3: a marker for the immunosuppressive capacity?

Multipotent mesenchymal stromal cells (MSCs) have immunosuppressive capacity but the exact mechanism by which they suppress proliferation of T lymphocytes is not fully understood. Recently, the characteristics and function of regulatory T lymphocytes (Tregs) have become better defined. Tregs and MSCs have immunosuppressive features in common. Here, we looked for a common basis for immunosuppression in these distinct cell types. Forkhead box P3 (FoxP3) and CD39 expression in MSCs was measured by flow cytometry and real-time quantitative polymerase chain reaction. The importance of FoxP3 in MSC-mediated immunosuppression was investigated by siRNA technology and mixed lymphocyte culture (MLC). The effect of 5-azacytidine and other immunosuppressive drugs on FoxP3 expression and immunosuppression by MSCs was explored by flow cytometry, MLC, and real-time quantitative polymerase chain reaction. MSCs express FoxP3 at variable levels, but they do not express CD39. FoxP3 MSCs suppress MLC to a greater extent than cells with lower FoxP3 expression. However, FoxP3-decreased MSCs were found to retain their immunosuppressive properties. 5-azacytitine had no effect on FoxP3 expression or MLC suppression by MSCs. However, immunosuppressive drugs led to increased FoxP3 levels and MLC inhibition in FoxP3 MSCs. This is the first demonstration of FoxP3 expression by MSCs. Although MSCs share several features with Tregs, and FoxP3 MSCs tend to be more immunosuppressive, MSCs do not require functional FoxP3 for their immunosuppressive activity. The increased MSC-mediated suppression of immune responses by immunosuppressive drugs deserves further investigation.

Growth differentiation factor 15: a prognostic marker for recurrence in colorectal cancer.

BACKGROUND: Growth differentiation factor 15 (GDF15) belongs to the transforming growth factor beta superfamily and has been associated with activation of the p53 pathway in human cancer. The aim of this study was to assess the prognostic value of GDF15 in patients with colorectal cancer (CRC). METHODS: Immunohistochemistry and tissue microarrays were used to analyse GDF15 protein expression in 320 patients with CRC. In a subgroup of 60 patients, the level of GDF15 protein in plasma was also measured using a solid-phase proximity ligation assay. RESULTS: Patients with CRC with moderate to high intensity of GDF15 immunostaining had a higher recurrence rate compared with patients with no or low intensity in all stages (stages I-III) (HR, 3.9; 95% CI, 1.16-13.15) and in stage III (HR, 10.32; 95% CI, 1.15-92.51). Patients with high plasma levels of GDF15 had statistically shorter time to recurrence (P=0.041) and reduced overall survival (P=0.002). CONCLUSION: Growth differentiation factor 15 serves as a negative prognostic marker in CRC. High expression of GDF15 in tumour tissue and high plasma levels correlate with an increased risk of recurrence and reduced overall survival.

The impact of inflammatory licensing on heme oxygenase-1-mediated induction of regulatory T cells by human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are characterized by their manifold immunomodulatory and regenerative properties. The stress-responsive, cytoprotective, and immunoregulatory molecule heme oxygenase-1 (HO-1) was recently identified as a key contributor for MSC-mediated suppression of alloactivated T cells. As HO-1 has also been implicated in the induction of regulatory T cells (Tregs), we sought to examine its impact on MSC-driven promotion of Tregs. Human MSCs were shown to induce, in a HO-1-dependent fashion, IL-10(+) Tr1 and transforming growth factor-ß(+) Th3 Treg-subsets in allo- and T-cell receptor-activated lymphocytes. Because inflammatory stimuli modulate ("license") human MSCs, we were interested in whether an in vitro alloreactive micro-milieu within mixed lymphocyte reactions (MLRs) alters the HO-1 expression. We observed a substantial down-regulation of HO-1 facilitated by yet unidentified soluble factor(s) produced in an MLR, and most probably occurring at the level of its major transcription-factor NF-E2-related factor 2. Interestingly, HO-1 lost its impact regarding suppressiveness, Treg induction, and promotion of IL-10 production for MSCs, which were prelicensed in an MLR environment. Taken together, we show that HO-1 produced by human MSCs beyond its direct suppressive function promotes formation of Tr1 and Th3 Tregs and IL-10 production, functions, which are taken over by other molecules, among them COX-2, after an alloreactive priming.

Dysphagia in adults operated on for esophageal atresia--use of a symptom score to evaluate correlated factors.

INTRODUCTION: Dysphagia is not unusual following repair of esophageal atresia (EA). The lack of a uniform definition has led to a variance when it comes to reporting the prevalence of dysphagia among patients operated on for EA. Our aim is to estimate the occurrence and degree of dysphagia, using a numerical score with its statistical versatility independent of a specific definition. The results are used to find early risk factors of dysphagia within this patient group. The results are also used to see whether we can find a correlation between dysphagia and symptoms of gastroesophageal reflux (GER) and quality of life (QoL). METHODS: 79 consecutive survivors operated on for EA in Gothenburg between 1968 and 1983 were located. Hospital charts were reviewed and patients received questionnaires on dysphagia, symptoms of GER and QoL. Dysphagia was measured by a numerical score, symptoms of GER were extracted using a predetermined questionnaire (GerdQ), and QoL was determined using the generic questionnaire SF-36. RESULTS: 73 patients (92.4%) returned the questionnaires. In order to make the study group as homogeneous as possible with regard to the malformation we choose to study the 63 patients representing the vast majority: those with Gross type C. 36 patients (57%) had symptoms of dysphagia to varying degrees. We did not find any aggravating factors in their hospital charts nor did we find any correlation to the most recent demographics. There was a significant difference in dysphagia scores when we compared Gross type C to the often more complex type A (p<0.05). We did not find any correlation to heartburn but a strong correlation to regurgitation with an OR of 2.8 (95% CI: 1.2-6.6). The QoL was good for this patient group, and we did not find any correlation between QoL and the dysphagia score. CONCLUSIONS: The dysphagia score provides easy-to-use results when it comes to evaluating the potential influence of dysphagia. Dysphagia is common within this patient group. Patients operated on for EA Gross type A seem to do worse when it comes to dysphagia. Regurgitation is associated with dysphagia, which could imply that GER is an aggravating factor. Further studies to support this finding will show whether there is a correlation between the dysphagia score and the results of 24-h pH-monitoring. If so, this could mean that treating GER might decrease dysphagia, at least in this patient group.

T cell recognition of HLA-A2 restricted tumor antigens is impaired by the oncogene HER2.

The HER2 oncogene is frequently over-expressed in human cancers and a promising target for immune therapy. Previous studies have shown that over-expression of mouse or rat HER2 leads to markedly reduced levels of major histocompatibility complex (MHC) class I and molecules of the antigen processing and presentation machinery (APM), thus resulting in a phenotype promoting tumor escape from the immune system. Our study focuses on analyzing the effect of HER2 on MHC class I antigen presentation and sensitivity to tumor-antigen specific cytotoxic T lymphocytes (CTLs) in HLA-A2.1(+) melanoma cell lines. We demonstrate significant inverse correlations both between the expression of HER2 and total MHC class I surface expression as well as between HER2 and HLA-A2. A significant reduction of HLA-A2 levels was found when melanoma and carcinoma cell lines were transfected with a human HER2 gene. A signaling-competent HER2 molecule was crucial for the observed HLA-A2 down-regulation, as transfectants expressing high levels of HER2 mutated in the tyrosine signaling domain did not show altered HLA-A2 expression. Importantly, the human melanoma cell line EST049 demonstrated reduced HER2 and melanoma antigen-specific recognition by CTLs upon HER2 transfection. In addition, high expression of HER2 prevented both IFN-γ mediated HLA-A2 up-regulation and improved recognition by HLA-A2-restricted CTLs in treated cells. Moreover, key APM molecules were down-regulated by HER2. These findings implicate that HER2 over-expressing tumors may be more prone to escape from HLA-A2 restricted CTLs suggesting that immunotherapy approaches inducing an integrated humoral, cellular and innate immune response would be most effective.

Increased thioredoxin-1 production in human naturally occurring regulatory T cells confers enhanced tolerance to oxidative stress.

Levels of regulatory T cells (Tregs) are increased in different cancer types as well as in inflammatory diseases, such as rheumatoid arthritis. Treg accumulation may result from aberrant proliferation and trafficking as well as greater resilience to oxidative stress compared with conventional T cells. This enhanced antioxidative capacity of Tregs possibly serves as feedback inhibition during inflammation and prevents uncontrolled immune reactions by favoring survival of suppressor rather than effector cells. In this study, we demonstrate that human Tregs express and secrete higher levels of thioredoxin-1, a major antioxidative molecule. Thioredoxin-1 has an essential role in maintaining their surface thiol density as the first line of antioxidative defense mechanisms and is sensitive to proinflammatory stimuli, mainly tumor necrosis factor-α, in a nuclear factor-κB-dependent fashion. The antiapoptotic and oncogenic potential of (secreted) Trx-1 suggests that it may exert effects in Tregs beyond redox regulation.

Mast cells generated from patients with atopic eczema have enhanced levels of granule mediators and an impaired Dectin-1 expression.

BACKGROUND: The disrupted skin barrier of patients with atopic eczema (AE) might facilitate contact between mast cells (MCs) in the skin and environmental triggers of the disease. One such trigger is the skin-colonizing yeast Malassezia sympodialis (M. sympodialis). In this study, we investigated the interaction of MC with M. sympodialis. METHODS: Mast cells were generated from peripheral blood CD34(+) progenitor cells of healthy controls (HC) and M. sympodialis-sensitized AE patients. Biopsy specimens were taken from HC and lesional AE skin for immunohistological stainings. RESULTS: The progenitor-derived MCs expressed the macrophage-inducible C-type lectin receptor Mincle, and exposure of these cells to M. sympodialis induced up-regulation of the mRNA expression of Mincle. Furthermore, we demonstrate that, when compared to HC, the progenitor-derived MCs from AE patients (i) contain more intrinsic granule mediators such as histamine, (ii) exhibit enhanced IL-6 release in response to M. sympodialis exposure, and (iii) have an impaired up-regulation of the fungal recognition receptor Dectin-1. In addition, analysis of skin sections from HC and AE patients revealed MCs as the predominant Dectin-1-expressing cell type in the skin. CONCLUSION: Our data indicate that progenitor-derived MCs from AE patients differ from those from HC. Further investigations with skin-derived MCs are necessary to confirm the observed differences which could provide new insights into the pathogenic mechanisms underlying AE.

Regulatory T cells in cancer.

At the present time, regulatory T cells (Tregs) are an integral part of immunology but the route from discovery of "suppressive" lymphocytes in the 1980s to the current established concept of Tregs almost 20 years later has been a rollercoaster ride. Tregs are essential for maintaining self-tolerance as defects in their compartment lead to severe autoimmune diseases. This vitally important function exists alongside the detrimental effects on tumor immunosurveillance and antitumor immunity. Beginning with the identification of CD4(+)CD25(+) Tregs in 1995, the list of Treg subsets, suppressive mechanisms, and knowledge about their various origins is steadily growing. Increase in Tregs within tumors and circulation of cancer patients, observed in early studies, implied their involvement in pathogenesis and disease progression. Several mechanisms, ranging from proliferation to specific trafficking networks, have been identified to account for their systemic and/or local accumulation. Since various immunotherapeutic approaches are being utilized for cancer therapy, various strategies to overcome the antagonistic effects exerted by Tregs are being currently explored. An overview on the biology of Tregs present in cancer patients, their clinical impact, and methods for modulating them is given in this review. Despite the extensive studies on Tregs in cancer many questions still remain unanswered. Even the paradigm that Tregs generally are disadvantageous for the control of malignancies is now under scrutiny. Insight into the specific role of Tregs in different types of neoplasias is the key for targeting them in a way that is beneficial for the clinical outcome.

Intratumoral forkhead box P3-positive regulatory T cells predict poor survival in cyclooxygenase-2-positive uveal melanoma.

BACKGROUND: Forkhead box P3 (FOXP3)-positive regulatory T cells (Tregs) are key mediators of peripheral tolerance and suppress efficient antitumor responses. Prostaglandin E(2) (PGE(2)) produced by inducible cyclooxygenase-2 (COX-2) can lead to Treg induction. COX-2 expression has been linked to tumorigenesis and growth in various malignancies. The objective of the current study was to investigate whether Tregs infiltrate uveal melanomas (UMs) and whether their prevalence is linked to COX-2 expression and the prediction of overall survival (OS). METHODS: One hundred patients who underwent enucleation after they were diagnosed with UM were included in the study. Immunohistochemical staining with monoclonal anti-FOXP3, anti-CD4, and anti-COX-2 antibodies was performed, and immunoreactivity was assessed. Correlations of COX-2 expression with the presence of Tregs, established clinicopathologic parameters, and OS were evaluated in univariate and multivariate analyses. RESULTS: High expression of COX-2 was predictive of shortened OS. FOXP3-positive Tregs were detectable in 24% of UMs and were restricted to malignant tissue. The extent of COX-2 expression was associated significantly with Treg prevalence (P = .004) and Treg intratumoral localization (P = .005). Intratumoral Tregs (but not the prevalence of Tregs) were independent marker for worse OS with a hazard ratio of 5.36 in patients with COX-2-positive tumors. CONCLUSIONS: The current results demonstrated that high COX-2 expression is associated with OS and Treg prevalence in UM. These findings are in line with the observations that COX-2/PGE(2) induces Tregs and that Tregs may alter antitumor responses, resulting in a negative effect on the clinical disease course. Intratumoral Tregs are an independent prognostic marker for COX-2-positive UM, and these results put COX-2 inhibitors and Treg depletion into the spotlight of potential novel treatment modalities for patients with UM.

Expression and prognostic significance of iNOS in uveal melanoma.

Uveal melanoma (UM) is the most common primary intraocular tumor in adults. Disease metastasis occurs in half of the patients and is uniformly fatal despite systemic therapy. Inducible nitric oxide synthase (iNOS) is associated with disease progression in various malignancies including cutaneous melanoma. In this retrospective cohort, we examined the prognostic value of iNOS in UM by performing immunohistochemistry on paraffin-embedded sections of primary tumors (90 patients) and matched primary and metastatic hepatic tumors (19 patients) with complete histopathological and clinical data. We show that iNOS is expressed in UM (57% of the patients) and high iNOS levels significantly (p = 0.04; hazard ratio (HR) = 2.3) predict disease-specific survival (DSS) as assessed by Kaplan-Meier analysis and univariate Cox's proportional hazards regression model. Furthermore, high iNOS expression in the UM primary tissue was significantly associated with metastatic disease and vice versa. Expression of iNOS in hepatic metastases significantly (p = 0.02) predicted a shortened survival as assessed by Kaplan-Meier analysis. However, iNOS did not appear to be a significant (p = 0.16; HR = 1.9) factor in the multivariate Cox's regression analysis performed together with the clinical parameters tumor diameter, tumor cell type, and tumor location in which only tumor diameter predicted DSS. In conclusion, iNOS predicts DSS in UM and may play a role in disease progression but it is not an independent prognostic factor.