Athanasia Mouzaki

BACKGROUND: In view of clinical observations and laboratory results that support a central role of the spleen in idiopathic thrombocytopenic purpura (ITP) pathophysiology, we studied the effect of splenectomy on type-1 and type-2 cytokine gene expression in an adult ITP case, refractory to conservative treatment. CASE PRESENTATION: The patient was subjected to splenectomy 9 months after the diagnosis with complete response, attaining platelet counts over 150 x 106/L within 10 days after the operation. Two consecutive blood samples were obtained from the patient, 3 and 7 months after the splenectomy for the purposes of this study. A control group consisted of 11 healthy adults. Peripheral blood mononuclear cells were prepared from each blood sample and cultured in vitro for 8 h with the addition of the mitogens phorbol myristate acetate and ionomycin. Total cellular RNA extracted from 106 cells was submitted to semiquantitave reverse transcriptase-polymerase chain reaction (RT-PCR) f...

Lung cancer is rarely cured by current therapeutic approaches. Although numerous studies have implicated FOXP3 positive regulatory T-cells in cancer pathogenesis, the role of FOXP3 in lung cancer pathogenesis remains unkown. Using immunohistochemistry FOXP3 expression was determined in 44 NSCLC tissue specimens, 20 samples from adjacent non neoplastic lung parenchyma and 5 normal lung tissue specimens. FOXP3 immunostaining was always nuclear in both tumor and non-neoplastic adjacent tissues. FOXP3 was also detected at lower levels in normal bronchial epithelium. Moreover, FOXP3 expression in cancer cells correlated with lymphocytic FOXP3-immunopositivity and the presence of lymph node metastasis. FOXP3 lymphocytic expression was also negatively associated with the age of the patients. FOXP3 is overexpressed in NSCLC cells and tumor-infiltrating lymphocytes. This study provides evidence that lymphocytic FOXP3 expression may be age related and that tumor FOXP3 expression is correlated...

The regulation of interleukin (IL)-2 gene expression has been investigated mainly in T lymphocytes, the predominant producers of IL-2. However, B cells can also synthesize IL-2. In the present study we analyzed the control of IL-2 promoter activity in Epstein-Barr virus (EBV)-transformed B cell clones which are capable of secreting IL-2 at a low level after stimulation with phorbol 12-myristate 13-acetate and the Ca2+ ionophore ionomycin. Transient transfections using reporter constructs with multiples of transcription factor binding sites from the IL-2 promoter [distal nuclear factor (NF)-AT, proximal NF-AT, AP-1/Octamer (UPS) or NF-ϰB (TCEd) sites] were performed. In EBV-transformed B clones, the ϰB site exerted the strongest inducible activity; the NF-AT binding sites showed either no or only weak activity compared to Jurkat T cells. An IL-2 promoter bearing a defective NF-ϰB site was completely inactive in EBV-B cells, white it still had activity in Jurkat T cells. In seven EBV-B cell clones or lines differing in their capacity to secrete IL-2, the activity of the IL-2 promoter correlated well with the status of IL-2 secretion. Similarly, a human immunodeficiency virus promoter, whose activity is controlled through ϰB factors, was found to be active in the IL-2-producing EBV-B cells, but inactive in the non-IL-2-producing cells. Electrophoretic mobility shift assays using protein extracts from EBV-B cells and the IL-2 NF-ϰB probe revealed the constitutive generation of ϰB complexes in IL-2-secreting cells consisting mainly of heterodimeric p50/p65 complexes. A weaker ϰB complex formation and faster-migrating complexes were detected in non-IL-2-secreting cells. These results demonstrate that the IL-2 NF-ϰB site is indispensable for the activity of the IL-2 promoter in EBV-transformed B cells, whereas other transcription factors appear to be less important for IL-2 expression in these cells.

Four new mouse monoclonal antibodies (mAb), ART-38, ART-35, ART-75 and ART-94, directed against the rat interleukin 2 receptor (IL 2R) have been developed. As shown by immunoprecipitation studies they all recognize specifically the 55-kDa subunit of the rat IL 2R. These mAb were compared to three previously characterized mouse mAb directed against the 55-kDa molecule of the rat IL 2R, namely the ART-18, ART-65 and OX-39 mAb. Out of all seven mAb, only ART-18 and OX-39 were found to inhibit the IL 2 binding to activated T cells, while IL 2R inhibited the binding of ART-18 alone. ART-18 was the only mAb found to inhibit the IL 2-dependent proliferation of cells carrying the IL 2R. Scatchard plot analyses showed gross differences in the numbers of mAb-binding sites ranging between 30000 (ART-65) and 165000 (ART-75) as well as in their affinities which ranged between 2.5 × 10−9 M (ART-38) and 8.3 × 10−10 M (OX-39). Cross inhibition studies revealed that the mAb recognize four different epitopes on the 55-kDa rat IL 2R subunit.

A case of Evans' syndrome with IgM deficiency and lymphopenia was studied before and after splenectomy. The lymphopenia was as a result of profound reduction of CD4 and CD8 cells. Study of cytokine secretion before splenectomy revealed a spontaneous Th1- and Th2-type cytokine production, and complete suppression of transforming growth factor (TGF)-β. After splenectomy, the patient achieved clinical remission, the natural killer (NK) cell number increased and the pattern of cytokine production showed normalization of interleukin (IL)-2, IL-4, IL-10, TGF-β and abolition of interferon (IFN)-γ production. We conclude that splenectomy had a beneficial effect owing to an increase in NK cells and an associated increase in TGF-β production.

Several studies have implicated leptin in the pathophysiology of neoplasias. We investigated the direct effect of leptin on malignant hematopoietic tissue that included: primary acute myeloid leukemia (AML) cells, leukemic cell lines and bone marrow biopsies from multiple myeloma (MM) patients. PBMC, T-cells, B-cells and monocytes from healthy subjects served as controls. We defined the patterns of OB-R isoform expression in AML cells and leukemic cell lines in comparison to control cells by RT-PCR. rLeptin upregulated the expression of OB-R and endogenous leptin in AML blasts and certain cell lines but not in control cells. Cytometric Bead Array analysis of pro- and anti-inflammatory cytokines showed that rleptin upregulates IL-6 secretion by AML cells, various cytokines by the leukemic cell lines tested and IL-10 secretion by control PBMC, contributed by monocytes. Western immunoblotting revealed that the effect of rleptin was independent of JAK-2/phospho-JAK-2 protein levels. Finally, MM biopsies stained positive for leptin and, to a lesser extend, OB-R. Immunoreactivity was confined mostly to the nucleus of the myeloma cells. Normal myelocytes, promyelocytes and megakaryocytes stained weakly positive, and erythroid cells were constantly negative. We propose that the leptin/OB-R system is strongly and directly involved in supporting the growth of hematopoietic malignancies.

Multiple myeloma (MM) remains an incurable disease by conventional therapy. MM tumor cells evade the immune system and can induce immunosuppression by producing immunomodifying agents such as TGF-beta, FasL, vascular endothelial growth factor and Muc-1. In the present study, we show that bone marrow cells from a patient suffering from MM IgG/k type, stage IIIA, when cultured, expressed granzyme B and perforin, normally expressed exclusively by cytotoxic T cells (CTLs) and natural killer (NK) cells. In addition, phenotypic analysis revealed that the cultured cells were activated antigen-presenting cells with NK targeting capacity. We propose that expression of these cytolytic enzymes may constitute an additional adoptive mechanism by the tumor cells to actively destroy the host immune effector cells.