Galectins in epithelial-mesenchymal transition: roles and mechanisms contributing to tissue repair, fibrosis and cancer metastasis.

Galectins are soluble glycan-binding proteins that interact with a wide range of glycoproteins and glycolipids and modulate a broad spectrum of physiological and pathological processes. The expression and subcellular localization of different galectins vary among tissues and cell types and change during processes of tissue repair, fibrosis and cancer where epithelial cells loss differentiation while acquiring migratory mesenchymal phenotypes. The epithelial-mesenchymal transition (EMT) that occurs in the context of these processes can include modifications of glycosylation patterns of glycolipids and glycoproteins affecting their interactions with galectins. Moreover, overexpression of certain galectins has been involved in the development and different outcomes of EMT. This review focuses on the roles and mechanisms of Galectin-1 (Gal-1), Gal-3, Gal-4, Gal-7 and Gal-8, which have been involved in physiologic and pathogenic EMT contexts.

Phosphatidic acid-PKA signaling regulates p38 and ERK1/2 functions in ligand-independent EGFR endocytosis.

Ligand-independent epidermal growth factor receptor (EGFR) endocytosis is inducible by a variety of stress conditions converging upon p38 kinase. A less known pathway involves phosphatidic acid (PA) signaling toward the activation of type 4 phosphodiesterases (PDE4) that decrease cAMP levels and protein kinase A (PKA) activity. This PA/PDE4/PKA pathway is triggered with propranolol used to inhibit PA hydrolysis and induces clathrin-dependent and clathrin-independent endocytosis, followed by reversible accumulation of EGFR in recycling endosomes. Here we give further evidence of this signaling pathway using biosensors of PA, cAMP, and PKA in live cells and then show that it activates p38 and ERK1/2 downstream the PKA inhibition. Clathrin-silencing and IN/SUR experiments involved the activity of p38 in the clathrin-dependent route, while ERK1/2 mediates clathrin-independent EGFR endocytosis. The PA/PDE4/PKA pathway selectively increases the EGFR endocytic rate without affecting LDLR and TfR constitute endocytosis. This selectiveness is probably because of EGFR phosphorylation, as detected in Th1046/1047 and Ser669 residues. The EGFR accumulates at perinuclear recycling endosomes colocalizing with TfR, fluorescent transferrin, and Rab11, while a small proportion distributes to Alix-endosomes. A non-selective recycling arrest includes LDLR and TfR in a reversible manner. The PA/PDE4/PKA pathway involving both p38 and ERK1/2 expands the possibilities of EGFR transmodulation and interference in cancer.

Galectin-8 mediates fibrogenesis induced by cyclosporine in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: During cyclosporine-induced gingival overgrowth, the homeostatic balance of gingival connective tissue is disrupted leading to fibrosis. Galectins are glycan-binding proteins that can modulate a variety of cellular processes including fibrosis in several organs. Here, we study the role of galectin-8 (Gal-8) in the response of gingival connective tissue cells to cyclosporine. METHODS: We used human gingival fibroblasts and mouse NIH3T3 cells treated with recombinant Gal-8 and/or cyclosporine for analyzing specific mRNA and protein levels through immunoblot, real-time polymerase chain reaction, ELISA and immunofluorescence, pull-down with Gal-8-Sepharose for Gal-8-to-cell surface glycoprotein interactions, short hairpin RNA for Gal-8 silencing and Student's t test and ANOVA for statistical analysis. RESULTS: Galectin-8 stimulated type I collagen and fibronectin protein levels and potentiated CTGF protein levels in TGF-ß1-stimulated human gingival fibroblasts. Gal-8 interacted with α5ß1-integrin and type II TGF-ß receptor. Gal-8 stimulated fibronectin protein and mRNA levels, and this response was dependent on FAK activity but not Smad2/3 signaling. Cyclosporine and tumor necrosis factor alpha (TNF-α) increased Gal-8 protein levels. Finally, silencing of galectin-8 in NIH3T3 cells abolished cyclosporine-induced fibronectin protein levels. CONCLUSION: Taken together, these results reveal for the first time Gal-8 as a fibrogenic stimulus exerted through ß1-integrin/FAK pathways in human gingival fibroblasts, which can be triggered by cyclosporine. Further studies should explore the involvement of Gal-8 in human gingival tissues and its role in drug-induced gingival overgrowth.

Scavenger Receptor-A deficiency impairs immune response of microglia and astrocytes potentiating Alzheimer's disease pathophysiology.

Late onset Alzheimer disease's (LOAD) main risk factor is aging. Although it is not well known which age-related factors are involved in its development, evidence points out to the involvement of an impaired amyloid-ß (Aß) clearance in the aged brain among possible causes. Glial cells are the main scavengers of the brain, where Scavenger Receptor class A (SR-A) emerges as a relevant player in AD because of its participation in Aß uptake and in the modulation of glial cell inflammatory response. Here, we show that SR-A expression is reduced in the hippocampus of aged animals and APP/PS1 mice. Given that Aß deposition increases in the aging brain, we generated a triple transgenic mouse, which accumulates Aß and is knockout for SR-A (APP/PS1/SR-A-/-) to evaluate Aß accumulation and the inflammatory outcome of SR-A depletion in the aged brain. The lifespan of APP/PS1/SR-A-/- mice was greatly reduced, accompanied by a 3-fold increase in plasmatic pro-inflammatory cytokines, and reduced performance in a working memory behavioral assessment. Microglia and astrocytes lacking SR-A displayed impaired oxidative response and nitric oxide production, produced up to 7-fold more pro-inflammatory cytokines and showed a 12-fold reduction in anti-inflammatory cytokines release, with conspicuous changes in lipopolysaccharide-induced glial activation. Isolated microglia from young and adult mice lacking SR-A showed a 50% reduction in phagocytic activity. Our results indicate that reduced expression of SR-A can deregulate glial inflammatory response and potentiate Aß accumulation, two mechanisms that could contribute to AD progression.

The Pediatric Choroidal and Ciliary Body Melanoma Study: A Survey by the European Ophthalmic Oncology Group.

PURPOSE: To collect comprehensive data on choroidal and ciliary body melanoma (CCBM) in children and to validate hypotheses regarding pediatric CCBM: children younger than 18 years, males, and those without ciliary body involvement (CBI) have more favorable survival prognosis than young adults 18 to 24 years of age, females, and those with CBI. DESIGN: Retrospective, multicenter observational study. PARTICIPANTS: Two hundred ninety-nine patients from 24 ocular oncology centers, of whom 114 were children (median age, 15.1 years; range, 2.7-17.9 years) and 185 were young adults. METHODS: Data were entered through a secure website and were reviewed centrally. Survival was analyzed using Kaplan-Meier analysis and Cox proportional hazards regression. MAIN OUTCOME MEASURES: Proportion of females, tumor-node-metastasis (TNM) stage, cell type, and melanoma-related mortality. RESULTS: Cumulative frequency of having CCBM diagnosed increased steadily by 0.8% per year of age between 5 and 10 years of age and, after a 6-year transition period, by 8.8% per year from age 17 years onward. Of children and young adults, 57% and 63% were female, respectively, which exceeded the expected 51% among young adults. Cell type, known for 35% of tumors, and TNM stage (I in 22% and 21%, II in 49% and 52%, III in 30% and 28%, respectively) were comparable for children and young adults. Melanoma-related survival was 97% and 90% at 5 years and 92% and 80% at 10 years for children compared with young adults, respectively (P = 0.013). Males tended to have a more favorable survival than females among children (100% vs. 85% at 10 years; P = 0.058). Increasing TNM stage was associated with poorer survival (stages I, II, and III: 100% vs. 86% vs. 76%, respectively; P = 0.0011). By multivariate analysis, being a young adult (adjusted hazard rate [HR], 2.57), a higher TNM stage (HR, 2.88 and 8.38 for stages II and III, respectively), and female gender (HR, 2.38) independently predicted less favorable survival. Ciliary body involvement and cell type were not associated with survival. CONCLUSIONS: This study confirms that children with CCBM have a more favorable survival than young adults 18 to 25 years of age, adjusting for TNM stage and gender. The association between gender and survival varies between age groups.

Psychosocial impact of prognostic genetic testing in the care of uveal melanoma patients: protocol of a controlled prospective clinical observational study.

BACKGROUND: Uveal melanoma patients with a poor prognosis can be detected through genetic analysis of the tumor, which has a very high sensitivity. A large number of patients with uveal melanoma decide to receive information about their individual risk and therefore routine prognostic genetic testing is being carried out on a growing number of patients. It is obvious that a positive prediction for recidivism in the future will emotionally burden the respective patients, but research on the psychosocial impact of this innovative method is lacking. The aim of the current study is therefore to investigate the psychosocial impact (psychological distress and quality of life) of prognostic genetic testing in patients with uveal melanoma. DESIGN AND METHODS: This study is a non-randomized controlled prospective clinical observational trial. Subjects are patients with uveal melanoma, in whom genetic testing is possible. Patients who consent to genetic testing are allocated to the intervention group and patients who refuse genetic testing form the observational group. Both groups receive cancer therapy and psycho-oncological intervention when needed. The psychosocial impact of prognostic testing is investigated with the following variables: resilience, social support, fear of tumor progression, depression, general distress, cancer-specific and general health-related quality of life, attitude towards genetic testing, estimation of the perceived risk of metastasis, utilization and satisfaction with psycho-oncological crisis intervention, and sociodemographic data. Data are assessed preoperatively (at initial admission in the clinic) and postoperatively (at discharge from hospital after surgery, 6-12 weeks, 6 and 12 months after initial admission). Genetic test results are communicated 6-12 weeks after initial admission to the clinic. DISCUSSION: We created optimal conditions for investigation of the psychosocial impact of prognostic genetic testing. This study will provide information on the course of disease and psychosocial outcomes after prognostic genetic testing. We expect that empirical data from our study will give a scientific basis for medico-ethical considerations.

Galectin-8 promotes migration and proliferation and prevents apoptosis in U87 glioblastoma cells.

BACKGROUND: Glioblastoma is one of the most aggressive cancers of the brain. Malignant traits of glioblastoma cells include elevated migration, proliferation and survival capabilities. Galectins are unconventionally secreted glycan-binding proteins that modulate processes of cell adhesion, migration, proliferation and apoptosis by interacting with beta-galactosides of cell surface glycoproteins and the extracellular matrix. Galectin-8 is one of the galectins highly expressed in glioblastoma cells. It has a unique selectivity for terminally sialylated glycans recently found enhanced in these highly malignant cells. A previous study in glioblastoma cell lines reported that Gal-8 coating a plastic surface stimulates two-dimensional motility. Because in other cells Gal-8 arrests proliferation and induces apoptosis, here we extend its study by analyzing all of these processes in a U87 glioblastoma cell model. METHODS: We used immunoblot and RT-PCR for Gal-8 expression analysis, recombinant Gal-8 produced in a bacteria system for Gal-8 treatment of the cells, and shRNA in lentivirus transduction for Gal-8 silencing. Cell migration as assessed in transwell filters. Cell proliferation, cell cycle and apoptosis were analyzed by FACS. RESULTS: Gal-8 as a soluble stimulus triggered chemotactic migration of U87 cells across the polycarbonate filter of transwell chambers, almost as intensively as fetal bovine serum. Unexpectedly, Gal-8 also enhanced U87 cell growth. Co-incubation of Gal-8 with lactose, which blocks galectin-glycan interactions, abrogated both effects. Immunoblot showed Gal-8 in conditioned media reflecting its secretion. U87 cells transduced with silencing shRNA in a lentiviral vector expressed and secreted 30-40 % of their normal Gal-8 levels. These cells maintained their migratory capabilities, but decreased their proliferation rate and underwent higher levels of apoptosis, as revealed by flow cytometry analysis of cell cycle, CFSE and activated caspase-3 staining. Proliferation seemed to be more sensitive than migration to Gal-8 expression levels. CONCLUSIONS: Gal-8, either secreted or exogenously enriched in the media, and acting through extracellular glycan interactions, constitutes a strong stimulus of directional migration in glioblastoma U87 cells and for the first time emerges as a factor that promotes proliferation and prevents apoptosis in cancerous cells. These properties could potentially contribute to the exaggerated malignancy of glioblastoma cells.

Galectin-8 binds to LFA-1, blocks its interaction with ICAM-1 and is counteracted by anti-Gal-8 autoantibodies isolated from lupus patients.

Galectin-8 belongs to a family of mammalian lectins that recognize glycoconjugates present on different cell surface components and modulate a variety of cellular processes. A role of Gal-8 in the immune system has been proposed based on its effects in immune cells, including T and B lymphocytes, as well as the presence of anti-Gal-8 autoantibodies in the prototypic autoimmune disease systemic lupus erythematosus (SLE). We have previously described that Gal-8 induces apoptosis in activated T cells interacting with certain ß1 integrins and this effect is counteracted by the anti-Gal-8 autoantibodies. Given that Gal-8 can potentially interact with several glycoproteins, here we analyzed the ß2 integrin Lymphocyte Function-Associated Antigen-1 (LFA-1), which is involved in leukocyte cell adhesion and immunological synapses. We show by GST-pull down assays that Gal-8 interacts with LFA-1 and this interaction is inhibited by anti-Gal-8 autoantibodies isolated from SLE patients. In cell adhesion assays, Gal-8 precluded the interaction of LFA-1 with its ligand Intracellular Adhesion Molecule-1 (ICAM-1). These results suggest that Gal-8 can exert immunosuppressive action not only by inducing apoptosis in activated T cells but also by negatively modulating the crucial function of LFA-1 in the immune system, while function-blocking autoantibodies counteract these effects.

D-Propranolol Impairs EGFR Trafficking and Destabilizes Mutant p53 Counteracting AKT Signaling and Tumor Malignancy.

Cancer therapy may be improved by the simultaneous interference of two or more oncogenic pathways contributing to tumor progression and aggressiveness, such as EGFR and p53. Tumor cells expressing gain-of-function (GOF) mutants of p53 (mutp53) are usually resistant to EGFR inhibitors and display invasive migration and AKT-mediated survival associated with enhanced EGFR recycling. D-Propranolol (D-Prop), the non-beta blocker enantiomer of propranolol, was previously shown to induce EGFR internalization through a PKA inhibitory pathway that blocks the recycling of the receptor. Here, we first show that D-Prop decreases the levels of EGFR at the surface of GOF mutp53 cells, relocating the receptor towards recycling endosomes, both in the absence of ligand and during stimulation with high concentrations of EGF or TGF-α. D-Prop also inactivates AKT signaling and reduces the invasive migration and viability of these mutp53 cells. Unexpectedly, mutp53 protein, which is stabilized by interaction with the chaperone HSP90 and mediates cell oncogenic addiction, becomes destabilized after D-Prop treatment. HSP90 phosphorylation by PKA and its interaction with mutp53 are decreased by D-Prop, releasing mutp53 towards proteasomal degradation. Furthermore, a single daily dose of D-Prop reproduces most of these effects in xenografts of aggressive gallbladder cancerous G-415 cells expressing GOF R282W mutp53, resulting in reduced tumor growth and extended mice survival. D-Prop then emerges as an old drug endowed with a novel therapeutic potential against EGFR- and mutp53-driven tumor traits that are common to a large variety of cancers.

Galectins in epithelial-mesenchymal transition: roles and mechanisms contributing to tissue repair, fibrosis and cancer metastasis

Galectins are soluble glycan-binding proteins that interact with a wide range of glycoproteins and glycolipids and modulate a broad spectrum of physiological and pathological processes. The expression and subcellular localization of different galectins vary among tissues and cell types and change during processes of tissue repair, fibrosis and cancer where epithelial cells loss differentiation while acquiring migratory mesenchymal phenotypes. The epithelial-mesenchymal transition (EMT) that occurs in the context of these processes can include modifications of glycosylation patterns of glycolipids and glycoproteins affecting their interactions with galectins. Moreover, overexpression of certain galectins has been involved in the development and different outcomes of EMT. This review focuses on the roles and mechanisms of Galectin-1 (Gal-1), Gal-3, Gal-4, Gal-7 and Gal-8, which have been involved in physiologic and pathogenic EMT contexts.

GALECTIN-8 Is a Neuroprotective Factor in the Brain that Can Be Neutralized by Human Autoantibodies.

Galectin-8 (Gal-8) is a glycan-binding protein that modulates a variety of cellular processes interacting with cell surface glycoproteins. Neutralizing anti-Gal-8 antibodies that block Gal-8 functions have been described in autoimmune and inflammatory disorders, likely playing pathogenic roles. In the brain, Gal-8 is highly expressed in the choroid plexus and accordingly has been detected in human cerebrospinal fluid. It protects against central nervous system autoimmune damage through its immune-suppressive potential. Whether Gal-8 plays a direct role upon neurons remains unknown. Here, we show that Gal-8 protects hippocampal neurons in primary culture against damaging conditions such as nutrient deprivation, glutamate-induced excitotoxicity, hydrogen peroxide (H2O2)-induced oxidative stress, and ß-amyloid oligomers (Aßo). This protective action is manifested even after 2 h of exposure to the harmful condition. Pull-down assays demonstrate binding of Gal-8 to selected ß1-integrins, including α3 and α5ß1. Furthermore, Gal-8 activates ß1-integrins, ERK1/2, and PI3K/AKT signaling pathways that mediate neuroprotection. Hippocampal neurons in primary culture produce and secrete Gal-8, and their survival decreases upon incubation with human function-blocking Gal-8 autoantibodies obtained from lupus patients. Despite the low levels of Gal-8 expression detected by real-time PCR in hippocampus, compared with other brain regions, the complete lack of Gal-8 in Gal-8 KO mice determines higher levels of apoptosis upon H2O2 stereotaxic injection in this region. Therefore, endogenous Gal-8 likely contributes to generate a neuroprotective environment in the brain, which might be eventually counteracted by human function-blocking autoantibodies.

Galectin-8 induces partial epithelial-mesenchymal transition with invasive tumorigenic capabilities involving a FAK/EGFR/proteasome pathway in Madin-Darby canine kidney cells.

Epithelial cells can acquire invasive and tumorigenic capabilities through epithelial-mesenchymal-transition (EMT). The glycan-binding protein galectin-8 (Gal-8) activates selective ß1-integrins involved in EMT and is overexpressed by certain carcinomas. Here we show that Gal-8 overexpression or exogenous addition promotes proliferation, migration, and invasion in nontumoral Madin-Darby canine kidney (MDCK) cells, involving focal-adhesion kinase (FAK)-mediated transactivation of the epidermal growth factor receptor (EGFR), likely triggered by α5ß1integrin binding. Under subconfluent conditions, Gal-8-overexpressing MDCK cells (MDCK-Gal-8H) display hallmarks of EMT, including decreased E-cadherin and up-regulated expression of vimentin, fibronectin, and Snail, as well as increased ß-catenin activity. Changes related to migration/invasion included higher expression of α5ß1 integrin, extracellular matrix-degrading MMP13 and urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) protease systems. Gal-8-stimulated FAK/EGFR pathway leads to proteasome overactivity characteristic of cancer cells. Yet MDCK-Gal-8H cells still develop apical/basolateral polarity reverting EMT markers and proteasome activity under confluence. This is due to the opposite segregation of Gal-8 secretion (apical) and ß1-integrins distribution (basolateral). Strikingly, MDCK-Gal-8H cells acquired tumorigenic potential, as reflected in anchorage-independent growth in soft agar and tumor generation in immunodeficient NSG mice. Therefore, Gal-8 can promote oncogenic-like transformation of epithelial cells through partial and reversible EMT, accompanied by higher proliferation, migration/invasion, and tumorigenic properties.

T-helper type 1 cytokine release is enhanced by in vitro zinc supplementation due to increased natural killer cells.

OBJECTIVE: We examined the influence of zinc on T-helper type 1 (Th1)/T-helper type 2 (Th2) balance in human lymphocytes. METHODS: Human peripheral blood mononuclear cells or diluted whole blood were cultured for 8 d in the presence of zinc (30 or 60 microM) or 1 microM of N, N, N', N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) (a zinc-specific chelator). Phytohemagglutinin-induced cytokine release was measured by enzyme-linked immunosorbent assay, and expression of CD56/CD69, CCR4/CD3, and CCR5/CD3 and intracellular labile zinc were detected by flow cytometry. RESULTS: We found that our in vitro supplementation resulted in an increase of intracellular labile zinc comparable to that of a 7-wk administration of 10 mg of zinc per day in vivo. Zinc triggered interferon-gamma release and impaired interleukin-10 release. Phenotypically, a Th2/Th1 shift could not be confirmed after detecting the Th1-specific chemokine receptor CCR5 or CCR4 for Th2 cells. Surprisingly, we detected a larger amount of CD56+ cells after zinc stimulation, leading us to the conclusion that the amount of interferon-gamma release after zinc supplementation might be attributed to the upregulation of natural killer cells after in vitro zinc supplementation rather than to a Th2/Th1 shift. CONCLUSION: We suggest that a nutritional intake of 10 mg of zinc increases the quantity of interferon-gamma-producing natural killer cells and strengthens the immune system against neoplasms and viral infections.

Suspected vitreous seeding of uveal melanoma: relevance of diagnostic vitrectomy.

AIM: To review all cases of suspected vitreous seeding of treated or untreated uveal melanoma at our clinic and to compare clinical, cytological and histological findings with patients' survival. METHODS: Retrospective non-randomised study of 23 patients with consecutive uveal melanoma who underwent diagnostic vitrectomy in our clinic between January 2000 and November 2013. Reason for vitrectomy was suspected dissemination of tumour cells inside the eye. Treated as well as treatment-naïve primary uveal melanomas were included in this study. Follow-up data of all patients were collected. RESULTS: The study included 23 patients with a mean age of 66 years. Four patients presented pigmented vitreous debris at initial presentation prior to treatment of the uveal melanoma. All but one of these four patients has been enucleated as a consequence of cytology-proven vitreous spreading of vital melanoma cells. The remaining 19 patients presented pigmented vitreous debris at a mean of 60 months following local tumour treatment. Thirteen of these patients had been treated with a ruthenium plaque (mean scleral dose 1295 Gy, mean apex dose 152 Gy), three with binuclid plaque (mean scleral dose 1005 Gy, mean apex dose 70 Gy) and three with proton beam radiation. Of the 19 patients, 10 showed only melanophages in the vitreous specimen, while the remaining 9 patients had vital tumour cells in vitreous cytology. Four out of these nine patients have been enucleated in the course of follow-up. During follow-up of our cohort of 23 patients, 4 patients died, but only 1 of them due to metastatic disease. CONCLUSION: The outcome of this small cohort study shows that obtaining a vitreous specimen helps to distinguish melanophages from vital tumour cells. We could not observe an increased risk of metastasis in patients who showed melanoma cell dissemination inside the eye, compared with those patients only showing melanophages. We therefore suggest to carefully re-evaluate the necessity of enucleation in every patient.

Comparing the Prognostic Value of BAP1 Mutation Pattern, Chromosome 3 Status, and BAP1 Immunohistochemistry in Uveal Melanoma.

Uveal melanoma (UM), a tumor of the eye, can be divided into 2 major classes correlating with patients' prognosis. Gene expression profiles and chromosome 3 status are correlated with tumor classification and prognosis. Somatic BAP1 mutations are another feature largely restricted to metastatic UM. Here we performed thorough BAP1 mutation analysis including sequencing and gene dosage analysis of all BAP1 coding exons as well as methylation analysis of the promoter CpG island in a set of 66 UMs. The results were compared with the BAP1 protein expression as determined by immunohistochemistry and the tumor-related survival of the patients. BAP1 sequencing and gene dosage analysis of BAP1 exons by multiplex ligation-dependent probe amplification revealed a mutation in 33 (89%) of 37 tumors with monosomy 3 (M3) or isodisomy 3. BAP1 mutations were not detected in any of the 28 tumors with disomy 3 or partial monosomy 3 (partM3). Most of the sequence mutations (21 of 28) were frame-shift, splice-site, or nonsense mutations leading to a premature termination codon. BAP1 protein as determined by immunohistochemistry was absent in all samples with a BAP1 mutation irrespective of the functional type of mutation. Kaplan-Meier analysis revealed a highly significant association between BAP1 protein staining and patients' survival (P=0.0004). The association between BAP1 mutation status and tumor-related survival was less pronounced but still significant (P=0.0023). We conclude that BAP1 protein staining is favorable over BAP1 mutation screening by Sanger sequencing for prognostic testing of UM patients.

Zinc supplementation induces regulatory T cells by inhibition of Sirt-1 deacetylase in mixed lymphocyte cultures.

SCOPE: Zinc is an essential trace element, regulating immune function. Its deficiency results in immune dysfunction and transplant rejection. In here, a benefit of zinc supplementation for the induction of tolerance was investigated, focusing on the TH 1-dominated allogeneic immune reaction. METHODS AND RESULTS: Allogeneic immune reaction was modeled by mixed lymphocyte culture (MLC). The effect of zinc supplementation was monitored via expression of cytokines and surface lineage markers using ELISA and flow cytometry. Epigenetic analyses were performed to investigate mechanisms underlying zinc-induced changes in regulatory T cell (Treg) activation. Results reveal that Tregs are induced when MLCs are treated with 50 µM zinc causing a decrease in IFNγ production. IL-2 and IL-10 expression were not affected. The teleology of this effect includes the inhibition of histone deacetylase Sirt-1-mediated Foxp3 deacetylation, resulting in its decreased degradation. CONCLUSION: In conclusion, zinc should be considered to prevent graft-versus-host disease (GVHD) as it is capable of stabilizing iTregs, resulting in increased numbers of this cell type while not suppressing the immune system.

Oncogene status as a diagnostic tool in ocular and cutaneous melanoma.

The majority of human tumours can be easily and correctly diagnosed based on clinical information and pathological assessment. In some cases however, correct diagnosis can prove difficult. In such cases, molecular approaches can be of significant diagnostic value. In recent years, the understanding of genetic alterations has greatly increased. In cutaneous melanoma, it is now well recognised, that 70-80% of tumours harbour BRAF and NRAS mutations. These mutations never occur in uveal melanoma. On the other hand activating GNAQ and GNA11 mutations are found in ∼90% of uveal melanomas, and are exceptionally rare in other melanomas (<1%). Here, we demonstrate a number of melanoma cases, where distinguishing if a tumour was of cutaneous or ocular origin was not possible based on clinical and pathological assessment. In these cases there was either atypical clinical presentation or metastasis of unclear primary. Histological distinction between uveal and cutaneous melanomas, especially at the stage of metastasis, is not reliable as they can be morphologically very similar. In all cases we present, a simple genetic assessment of oncogene mutation status was able to clearly define the melanoma type. This type of genetic assessment is of great diagnostic value and due to its simplicity could be performed in routine clinical practice even in smaller institutions.

Gender differences in adolescent depression: do symptoms differ for boys and girls?

BACKGROUND: Limited prior research suggests that depressed women are more likely to experience certain symptoms of depression than are depressed men. The purpose of this study was to examine whether such gender differences in depressive symptoms are present during adolescence. METHODS: The Childhood Version of the Schedule for Affective Disorders and Schizophrenia and the Beck Depression Inventory were administered to adolescents presenting for evaluation at an outpatient clinic (n=383; ages 11.9 to 20.0). RESULTS: Depressed girls and boys had similar symptom prevalence and severity ratings for most depressive symptoms. However, depressed girls had more guilt, body image dissatisfaction, self-blame, self-disappointment, feelings of failure, concentration problems, difficulty working, sadness/depressed mood, sleep problems, fatigue, and health worries than depressed boys on some comparisons. In contrast, depressed boys had higher clinician ratings of anhedonia, depressed morning mood, and morning fatigue. LIMITATIONS: Longitudinal research is needed to test whether such relatively gender-specific symptoms play different roles in the onset, maintenance, or remittance of depression for boys and girls. CONCLUSIONS: These findings indicate that, in general, the experience of depression is highly similar for adolescent girls and boys. However, some gender differences previously found among depressed adults appear to be present by adolescence, possibly suggesting somewhat distinct etiologies for depression among males and females.

Anti-ribosomal P protein autoantibodies from patients with neuropsychiatric lupus impair memory in mice.

OBJECTIVE: To define whether anti-ribosomal P (anti-P) autoantibodies from patients with neuropsychiatric systemic lupus erythematosus (NPSLE) impair the function of hippocampal neurons that express the neuronal surface P antigen (NSPA) when accessing the brain via circulating blood. METHODS: We used anti-P antibodies from patients with NPSLE and rabbit-generated anti-P and anti-NSPA antibodies. Primary hippocampal neurons from mice were analyzed to determine antibody cell surface binding (double immunofluorescence), intracellular calcium variations (Fura 2 AM), and apoptosis (caspase 3 activation). Hippocampal-dependent spatial flexible memory was assessed in mice subjected to a water maze test 24 hours after an intravenous injection of anti-P or anti-NSPA, using lipopolysaccharide (LPS) to permeate the blood-brain barrier. Presence of antibodies and apoptosis in the hippocampus was studied using immunohistochemistry and TUNEL assays. RESULTS: Hippocampal neurons expressed NSPA on the cell surface, as revealed by anti-P and anti-NSPA staining colocalization, and responded to both anti-P and anti-NSPA by exhibiting increased intracellular calcium levels. Neuronal apoptosis was induced when anti-P was directly injected by stereotaxis into the hippocampus or added to primary cultures. Upon LPS treatment, intravenously injected anti-P impaired memory but did not elicit neuronal apoptosis in the hippocampus, where it was detectable in low amounts. Anti-NSPA antibodies also impaired memory. CONCLUSION: Anti-P antibodies interact with NSPA on the surface of hippocampal neurons leading to apoptotic death or to functional perturbations, results that are likely dependent on the concentration of these antibodies. Circulating anti-P can access the hippocampus and impair memory without requiring neuronal death when the blood-brain barrier is disrupted. NSPA can mediate antibody-driven diffuse brain dysfunction, and anti-P might contribute to the cognitive impairment that is frequently observed in SLE.

Analysis of SDHD promoter mutations in various types of melanoma.

OBJECTIVES: Recently, recurrent mutations in regulatory DNA regions, such as promoter mutations in the TERT gene were identified in melanoma. Subsequently, Weinhold et al. reported SDHD promoter mutations occurring in 10% of melanomas and being associated with a lower overall survival rate. Our study analyzes the mutation rate and clinico-pathologic associations of SDHD promoter mutations in a large cohort of different melanoma subtypes. METHODS: 451 melanoma samples (incl. 223 non-acral cutaneous, 38 acral, 33 mucosal, 43 occult, 43 conjunctival and 51 uveal melanoma) were analyzed for the presence of SDHD promoter mutations by Sanger-sequencing. Statistical analysis was performed to screen for potential correlations of SDHD promoter mutation status with various clinico-pathologic criteria. RESULTS: The SDHD promoter was successfully sequenced in 451 tumor samples. ETS binding site changing SDHD promoter mutations were identified in 16 (4%) samples, of which 5 mutations had not been described previously. Additionally, 5 point mutations not located in ETS binding elements were identified. Mutations in UV-exposed tumors were frequently C>T. One germline C>A SDHD promoter mutation was identified. No statistically significant associations between SDHD promoter mutation status and various clinico-pathologic variables or overall patient survival were observed. CONCLUSIONS: Melanomas harbor recurrent SDHD promoter mutations, which occur primarily as C>T alterations in UV-exposed melanomas. In contrast to the initial report and promoter mutations in the TERT gene, our analysis suggests that SDHD promoter mutations are a relatively rare event in melanoma (4% of tumors) of unclear clinical and prognostic relevance.