Elizaveta Fasler-Kan

Normal Thymic Architecture and Negative Selection Are Associated with Aire Expression, the Gene Defective in the Autoimmune-Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy (APECED)

T cell development is tightly controlled by thymic stromal cells. Alterations in stromal architecture affect T cell maturation and the development of self-tolerance. The monogenic autoimmune syndrome APECED (autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy) is characterized by the loss of self-tolerance to multiple organs. Although mutations in the autoimmune regulator (AIRE) gene are responsible for this disease, the function of AIRE is not known. Here we report on the spatial and temporal pattern of murine Aire expression during thymic ontogeny and T cell selection. Early during development, thymic Aire transcription is critically dependent on RelB and occurs in epithelial cells in response to lymphocyte-mediated signals. In adult tissue, Aire expression is confined to the medulla and the corticomedullary junction, where it is modulated by thymocytes undergoing negative selection. Aire may determine thymic stromal organization and with it the induction of self-tolerance.

Circulating microparticles: square the circle

BackgroundThe present review summarizes current knowledge about microparticles (MPs) and provides a systematic overview of last 20 years of research on circulating MPs, with particular focus on their clinical relevance.ResultsMPs are a heterogeneous population of cell-derived vesicles, with sizes ranging between 50 and 1000 nm. MPs are capable of transferring peptides, proteins, lipid components, microRNA, mRNA, and DNA from one cell to another without direct cell-to-cell contact. Growing evidence suggests that MPs present in peripheral blood and body fluids contribute to the development and progression of cancer, and are of pathophysiological relevance for autoimmune, inflammatory, infectious, cardiovascular, hematological, and other diseases. MPs have large diagnostic potential as biomarkers; however, due to current technological limitations in purification of MPs and an absence of standardized methods of MP detection, challenges remain in validating the potential of MPs as a non-invasive and early diagnostic platform.ConclusionsImprovements in the effective deciphering of MP molecular signatures will be critical not only for diagnostics, but also for the evaluation of treatment regimens and predicting disease outcomes.

Defective Jak‐STAT signal transduction pathway in melanoma cells resistant to growth inhibition by interferon‐α

Advanced malignant melanoma is an aggressive malignancy with poor prognosis. Current therapeutic strategies have a modest success rate. The most promising treatment consists of a combination of chemotherapy with interferon‐α, but complete response rates remain less than 15%. Interferon‐α is also effective in adjuvant therapy for non‐advanced melanoma treated surgically. The molecular mechanisms leading to loss of growth restraints and gain of growth‐promoting functions during carcinogenesis of malignant melanoma are not understood in detail. Here, we studied 9 human melanoma cell lines with regard to growth inhibition by interferon‐α and defects in intracellular signal transduction through the Jak‐STAT pathway. In 3 cell lines, we found a complete loss of growth restraint by interferon‐α. In all of them, different components of the Jak‐STAT pathway were defective. Since signal transduction through the Jak‐STAT pathway is necessary for antiviral and antiproliferative effects of interferons, we conclude that defects in this pathway may be one of the mechanisms that lead to cancer progression through loss of growth‐restraining functions. Moreover, our results indicate that a subgroup of melanomas could be completely resistant to interferon‐α and should therefore not be treated with this cytokine. Int. J. Cancer 85:720–725, 2000.

Imaging Flow Cytometry: Coping with Heterogeneity in Biological Systems

Imaging flow cytometry (IFC) platforms combine features of flow cytometry and fluorescent microscopy with advances in data-processing algorithms. IFC allows multiparametric fluorescent and morphological analysis of thousands of cellular events and has the unique capability of identifying collected events by their real images. IFC allows the analysis of heterogeneous cell populations, where one of the cellular components has low expression (<0.03%) and can be described by Poisson distribution. With the help of IFC, one can address a critical question of statistical analysis of subcellular distribution of proteins in a cell. Here the authors review advantages of IFC in comparison with more traditional technologies, such as Western blotting and flow cytometry (FC), as well as new high-throughput fluorescent microscopy (HTFM), and discuss further developments of this novel analytical technique.

Cytokine signaling in the human brain capillary endothelial cell line hCMEC/D3

Brain microvascular endothelial cells are part of the blood-brain barrier and participate actively in immunological processes including cytokine-mediated inflammatory reactions. Using the human brain capillary endothelial cell line hCMEC/D3, activation of JAK/STAT signaling pathways were studied in response to stimulation by cytokines. The phenotype of hCMEC/D3 cells was confirmed by flow cytometry analysis of cell adhesion factors (cluster of differentiation molecules CD31 and CD34) and the von Willebrand factor endothelial marker was detected by immunofluorescence. Strong STAT1, STAT6 and STAT3 activation was observed in response to interferon-gamma (IFN-gamma), interleukin 4 (IL-4) and interleukin 6 (IL-6), respectively. Nuclear translocation of phosphorylated STAT proteins was visualized by confocal microscopy. Treatment of hCMEC/D3 cells with IFN-gamma resulted in interferon-induced upregulation of major histocompatibility complex (MHC) class I within 48h. Interferon-alpha (IFN-alpha) did not activate STAT1 or STAT3 nor did it induce MHC class I upregulation. Therefore, hCMEC/D3 cells were judged to be non-responsive to IFN-alpha. We also observed that hCMEC/D3 cells exhibit functional expression of alternative cytokine signal transduction pathways (i.e. TNF-alpha mediated activation of NF-kappaB). Together these results indicate that human blood-brain barrier hCMEC/D3 cells are responsive towards stimulation with various cytokines. We conclude that this unique cell line can be used to explore in vitro human blood-brain barrier functionality under proinflammatory conditions.

Serum hepcidin concentrations correlate with ferritin in patients with inflammatory bowel disease

BACKGROUND AND AIMS Anemia is a frequent complication of inflammatory bowel disease (IBD). Hepcidin, a key mediator in this anemia, is up-regulated by high iron levels and inflammation, and serum levels are elevated in IBD. However, the extent of inflammatory activity and iron deficiency for the regulation of hepcidin is not known. This study aimed to evaluate serum hepcidin levels in anemic and non-anemic IBD patients, with iron or non-iron deficiency, and active or inactive disease. METHODS This retrospective, observational study analyzed serum hepcidin levels from 247 patients with IBD (130 Crohns patients and 117 with ulcerative colitis) recruited at Swiss Inflammatory Bowel Disease Cohort Study centers. Patients were divided into 5 different groups using criteria of active and inactive diseases (C-reactive protein, and CDAI/MTWAI=disease activity-index), anemia (hemoglobin) and iron deficiency (ferritin) and compared to healthy controls with no signs of anemia and normal ferritin levels. Hepcidin was measured using enzyme-linked immunosorbent assay. RESULTS Independent of inflammatory activity, all patients with decreased ferritin (<30μg/L) had significantly lower hepcidin levels when compared to patients and healthy controls having normal ferritin (>30μg/L). A significant correlation between serum ferritin levels and serum hepcidin was found (Spearmans Rho=0.491; p<0.001). A backward multi-linear stepwise regression analysis showed that only ferritin, and none of the inflammatory markers or age and sex correlated significantly (p=0.005) with hepcidin. CONCLUSION This retrospective analysis suggests that iron deficiency is the key trigger for hepcidin regulation in IBD patients with anemia.

Activated STAT3 in Choroidal Neovascular Membranes of Patients with Age-Related Macular Degeneration

Purpose: The Jak/STAT (Janus tyrosine kinase/signal transducers and activators of transcription) pathway is critical for growth control, developmental regulation and homeostasis. Here we studied the expression of STAT proteins in the proliferative disease of age-related macular degeneration (AMD) with choroidal neovascular membranes (CNVM). The STATs are cytoplasmic proteins with roles as signal messengers and transcription factors that participate in normal cellular responses to cytokines and growth factors. Abnormal activity of certain STAT family members, particularly STAT3 and STAT5, is associated with a wide variety of human malignancies and other diseases. Here were studied STAT activation in CNVM of patients with AMD. Methods: Sections of formalin-fixed, paraffin-embedded samples from 8 eyes with AMD and 5 controls were included in this study. Immunohistochemical staining was performed using antibodies against activated STAT1, STAT3 and STAT5 proteins, and tenascin. Results: In CNVM, we observed a strong positive staining for tenascin and STAT3 in retinal pigmented epithelial (RPE) cells restricted to areas of developing scars. In contrast, STAT3 immunoreactivity failed in areas completely composed of fibrovascular disciform scar material. In addition, no immunoreactivity for both STAT1 and STAT5 was detected in all CNVM and in all control samples. Conclusion: In CNVM, activation of STAT3 appears in RPE cells simultaneously with the formation of scars.

Cell sorting in cancer research--diminishing degree of cell heterogeneity.

Increasing evidence of intratumor heterogeneity and its augmentation due to selective pressure of microenvironment and recent achievements in cancer therapeutics lead to the need to investigate and track the tumor subclonal structure. Cell sorting of heterogeneous subpopulations of tumor and tumor-associated cells has been a long established strategy in cancer research. Advancement in lasers, computer technology and optics has led to a new generation of flow cytometers and cell sorters capable of high-speed processing of single cell suspensions. Over the last several years cell sorting was used in combination with molecular biological methods, imaging and proteomics to characterize primary and metastatic cancer cell populations, minimal residual disease and single tumor cells. It was the principal method for identification and characterization of cancer stem cells. Analysis of single cancer cells may improve early detection of tumors, monitoring of circulating tumor cells, evaluation of intratumor heterogeneity and chemotherapeutic treatments. The aim of this review is to provide an overview of major cell sorting applications and approaches with new prospective developments such as microfluidics and microchip technologies.

The Tim-3-galectin-9 Secretory Pathway is Involved in the Immune Escape of Human Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is a severe and often fatal systemic malignancy. Malignant cells are capable of escaping host immune surveillance by inactivating cytotoxic lymphoid cells. In this work we discovered a fundamental molecular pathway, which includes ligand-dependent activation of ectopically expressed latrophilin 1 and possibly other G-protein coupled receptors leading to increased translation and exocytosis of the immune receptor Tim-3 and its ligand galectin-9. This occurs in a protein kinase C and mTOR (mammalian target of rapamycin)-dependent manner. Tim-3 participates in galectin-9 secretion and is also released in a free soluble form. Galectin-9 impairs the anti-cancer activity of cytotoxic lymphoid cells including natural killer (NK) cells. Soluble Tim-3 prevents secretion of interleukin-2 (IL-2) required for the activation of cytotoxic lymphoid cells. These results were validated in ex vivo experiments using primary samples from AML patients. This pathway provides reliable targets for both highly specific diagnosis and immune therapy of AML.

The immune receptor Tim-3 mediates activation of PI3 kinase/mTOR and HIF-1 pathways in human myeloid leukaemia cells.

The T-cell immunoglobulin and mucin domain 3 (Tim-3) is a plasma membrane-associated protein that is highly expressed in human acute myeloid leukaemia cells. As an acute myeloid leukaemia antigen, it could therefore be considered as a potential target for immune therapy and highly-specific drug delivery. However, a conceptual understanding of its biological role is required before consideration of this protein for therapeutic settings. Here, we reveal the detailed mechanism of action underlying the biological responses mediated by the Tim-3 receptor in myeloid cells. Our studies demonstrate that Tim-3 triggers growth factor type responses in acute myeloid leukaemia cells by activating a phosphatidylinositol-3 kinase (PI-3K)/mammalian target of rapamycin (mTOR) pathway. In addition, the receptor activates hypoxic signalling pathways upregulating glycolysis and pro-angiogenic responses. These findings suggest that Tim-3 could be used as a potential therapeutic target for immune therapy and drug delivery in human acute myeloid leukaemia cells.