Iskra Altankova

Naked DNA and adenoviral immunizations for immunotherapy of prostate cancer : A phase I/II clinical trial

Introduction and Objectives: Animal studies have indicated that the use of syngeneic dendritic cells that have been transfected ex vivo with DNA for tumor–specific antigen results in tumor regression and decreased number of metastases. Additional studies have also suggested the possibility to modulate the dendritic cells in vivo either by ‘naked’ DNA immunization or by injecting replication–deficient viral vectors that carry the tumor–specific DNA. Using the prostate– specific membrane antigen (PSMA) as a target molecule, we have initiated a clinical trial for immunotherapy of prostate cancer. The primary objective of the study was to determine the safety of the PSMA vaccine after repeated intradermal injections.Methods: We have included the extracellular human PSMA DNA as well as the human CD86 DNA into separate expression vectors (PSMA and CD86 plasmids), and into a combined PSMA/CD86 plasmid. In addition, the expression cassette from the PSMA plasmid was inserted into a replication deficient adenoviral expression vector. Twenty–six patients with prostate cancer were entered into a phase I/II toxicity–dose escalation study, which was initiated in spring 1998. Immunizations were performed intradermally at weekly intervals. Doses of DNA between 100 and 800 μg and of recombinant virus at 5×108 PFUs per application were used.Results and Conclusion: No immediate or long–term side effects following immunizations have been recorded. All patients who received initial inoculation with the viral vector followed by PSMA plasmid boosts showed signs of immunization as evidenced by the development of a delayed–type hypersensitivity reaction after the PSMA plasmid injection. In contrast, of the patients who received a PSMA plasmid and CD86 plasmid, only 50% showed signs of successful immunization. Of the patients who received PSMA plasmid and soluble GM–CSF, 67% were immunized. However, all patients who received the PSMA/CD86 plasmid and sGM–CSF became immunized. The patients who did not immunize during the first round were later successfully immunized after a boost with the viral vector. The heterogeneity of the medical status and the presence in many patients of concomitant hormone therapy does not permit unequivocal interpretation of the data with respect to the effectiveness of the therapy. However, several responders, as evidenced by a change in the local disease, distant metastases, and PSA levels, can be identified. A phase II clinical study to evaluate the effectiveness of the therapy is currently underway.

Mesenchymal stem cells from human bone marrow or adipose tissue differently modulate mitogen-stimulated B-cell immunoglobulin production in vitro.

Mesenchymal stem cells (MSC) have been characterized as multipotent cells which are able to differentiate into several mesodermal and nonmesodermal lineage cells and this feature along with their extensive growth and comprehensive immunomodulatory properties establish them as a promising tool for therapeutic applications, including cell‐based tissue engineering and treatment of immune‐mediated disorders. Although bone marrow (BM) is the most common MSC source, cells with similar characteristics have been shown to be present in several other adult tissues. Adipose tissue (AT), large quantities of which can be easily obtained, represents an attractive alternative to BM in isolating adipose tissue‐derived MSC (AT‐MSC). BM‐MSCs and AT‐MSCs share some immunomodulatory properties as they are both not inherently immunogenic and suppress the proliferation of alloantigen‐ or mitogen‐stimulated T‐cells. Our purpose was to comparatively examine under appropriate in vitro conditions, phenotypes, morphology and some functional properties of BM‐MSCs and AT‐MSCs, such as differentiation potential and especially the ability to suppress the immunoglobulin production by mitogen‐stimulated B‐cells. While the morphological, immunophenotypical, colony‐forming and adipogenic characteristics of both types of cells were almost identical, AT‐MSCs showed less potential for osteogenic differentiation than BM‐MSCs. We found that AT‐MSCs not only inhibited the Ig‐production but also suppressed this B‐cell function to a much greater extent compared to BM‐MSC. This finding supports the potential role of AT‐MSCs as an alternative to BM‐MSCs for clinical purposes.

Conditioned Medium from Adipose Tissue-Derived Mesenchymal Stem Cells Induces CD4+FOXP3+ Cells and Increases IL-10 Secretion

Mesenchymal stem cells (MSCs) are a new and promising tool for therapy of autoimmune disorders. In recent years their possibility to take part in the modulation of the immune response is discussed. The exact mechanisms for immunoregulation realized by MSCs are not clear yet, but interactions with other immunoregulatory cells may be involved in this process. The investigation of the influence of MSCs on the expression of FoxP3 and cytokine secretion by T helper cells was the aim of this study. T helper cells were isolated from PBMCs by magnetic separation and MSCs were isolated from human adipose tissue, and CD4+ T cells were cultured with conditional medium of MSCs. The methods which were used include flow cytometry, ELISA, and Human Proteome profiler kits. The results demonstrated that secretory factors in MSCs conditional medium lead to increased expression of FoxP3 and increased secretion of IL-10 by T helpers. The obtained results give us opportunity to discuss the interaction between two kinds of immunoregulatory cells: MSCs and FoxP3+ T helpers. We suppose that this interaction leads to increased number of immunosuppressive helpers which secrete IL-10. MSCs provide some of their immunosuppressive functions acting on T regulatory cells, and we believe that IL-6 secreted by MSCs is involved in this process.

CD83+ Monocyte-Derived Dendritic Cells are Present in Human Decidua and Progesterone Induces Their Differentiation In Vitro

Problem:  Dendritic cells (DCs) play an important role in antigen presentation and immunoregulation. Modifications of the immune response during pregnancy require the participation of DC. The aim of this study was to follow‐up the changes of DCs in human decidua and their correlations to progesterone (Pg) concentrations.

Female sex steroid hormones modify some regulatory properties of monocyte-derived dendritic cells.

Monocyte‐derived dendritic cells (mDCs) are present in human decidua during the first month of pregnancy where they experience the effect of the increased concentration of progesterone (Pg) and estradiol (Estr). The aim of our study was to assess the effect of these reproductive hormones on the immunomodulatory role of mDCs.

Dynamics of membrane translocation of phosphatidylserine during apoptosis detected by a monoclonal antibody.

Translocation of phosphatidylserine (PS) to the outer leaflet of the cellular membrane seems to be a key step in apoptosis and cell activation. In this paper, the production and characterization of a monoclonal antibody designated as Mab 1H6 is described which does not show cross reactivity with others anionic phospholipids. It is demonstrated that Mab1H6 can recognize externalized PS at early stages after the induction of apoptosis shown by both flow cytometry and immunofluorescence. Our results show that translocation of PS can be detected as early as 5 min by immunofluorescence and 10 min by flow cytometry after the treatment of cells and a specific dynamics is observed concerning the location and distribution of the staining. These data prove that antibody Mab 1H6 can be used as a specific probe for detection of PS translocation.

Human dendritic cells genetically engineered to express cytosolically retained fragment of prostate-specific membrane antigen prime cytotoxic T-cell responses to multiple epitopes

The ability of two plasmid DNA vaccines to stimulate lymphocytes from normal human donors and to generate antigen-specific responses is demonstrated. The first vaccine (truncated; tPSMA) encodes for only the extracellular domain of prostate-specific membrane antigen (PSMA). The product, expressed following transfection with this vector, is retained in the cytosol and degraded by the proteasomes. For the “secreted” (sPMSA) vaccine, a signal peptide sequence is added to the expression cassette and the expressed protein is glycosylated and directed to the secretory pathway. Monocyte-derived dendritic cells (DCs) are transiently transfected with either sPSMA or tPSMA plasmids. The DCs are then used to activate autologous lymphocytes in an in vitro model of DNA vaccination. Lymphocytes are boosted following priming with transfected DCs or with peptide-pulsed monocytes. Their reactivity is tested against tumor cells or peptide-pulsed T2 target cells. Both tPSMA DCs and sPSMA DCs generate antigen-specific cytotoxic T-cell responses. The immune response is restricted toward one of the four PSMA-derived epitopes when priming and boosting is performed with sPSMA. In contrast, tPSMA-transfected DCs prime T cells toward several PSMA-derived epitopes. Subsequent repeated boosting with transfected DCs, however, restricts the immune response to a single epitope due to immunodominance.

Depletion of CD25+ cells from human T-cell enriched fraction eliminates immunodominance during priming with dendritic cells genetically modified to express a secreted protein

The ability of dendritic cells (DCs), genetically modified with one of two types of plasmid DNA vaccines to stimulate lymphocytes from normal human donors and to generate antigen-specific responses, is compared. The first type, also called “secreted” vaccine (sVac), encodes for the full length of the human prostate-specific antigen (PSA) with a signal peptide sequence so that the expressed product is glycosylated and directed to the secretory pathway. The second type, truncated vaccines (tVacs), encodes for either hPSA or human prostate acidic phosphatase (hPAP), both of which lack signal peptide sequences and are retained in the cytosol and degraded by the proteasomes following expression. Monocyte-derived dendritic cells are transiently transfected with either sVac or one of two tVacs. The DCs are then used to activate CD25+-depleted or nondepleted autologous lymphocytes in an in vitro model of DNA vaccination. Lymphocytes are boosted following priming with transfected DCs, peptide-pulsed DCs or monocytes. Their reactivity is tested against tumor cells or peptide-pulsed T2 target cells. Both tVacDCs and sVacDCs generate antigen-specific cytotoxic T-cell responses. The immune response is restricted towards one of the three antigen-derived epitopes when priming and boosting is performed with sVacDCs. In contrast, tVac-transfected DCs prime T cells towards all antigen-derived epitopes. Subsequent repeated boosting with transfected DCs, however, restricts the immune response to a single epitope due to immunodominance. While CD25+ cell depletion prior to priming with sVacDCs alleviates immunodominance, cotransfection of dendritic cells with GITR-L does so in some but not all cases.

Alterations in cytokine gene expression profile in colon mucosa of Inflammatory Bowel Disease patients on different therapeutic regimens

HIGHLIGHTSFoxP3 and IL‐6 gene expression were significantly higher in the inflamed IBD mucosa.IBD forms differ in expression of IL‐17A (UC) and TGF&bgr;1 (CD) in inflamed mucosa.There were mild differences between the adjacent “normal” IBD and healthy mucosa.The serum levels of IL‐23, TGF&bgr;1 and IL‐6 were higher in IBD patients.Immunosuppressants are more beneficial for the immune tolerance in IBD than 5‐ASA. ABSTRACT Inflammatory bowel disease (IBD) is assumed to be caused by genetic and environmental factors that interact together in promoting intestinal immune dysregulation where cytokines have validated role. However, the underlying intimate mechanisms in the human IBD involving cytokines still needs to be supplemented especially in the clinical context. The aim of this study was to investigate the expression of some inflammatory and regulatory cytokines (IL‐17A, IL‐23, IL‐6, TGF&bgr;1, and IL‐10) as well as of the transcription factor FoxP3 in mucosal samples of IBD and non‐IBD patients. We assessed the mRNA relative quantities (RQ) of the above‐mentioned cytokines and the transcription factor FoxP3 in paired colonic samples (inflamed and adjacent normal mucosa) from 37 patients with IBD and in normal mucosal tissue in 12 persons without IBD by performing a qRT‐PCR assay and tested the protein levels of target cytokines in serum samples. The patients were divided into three groups: without any therapy (n = 10), on 5‐ASA (n = 11) and on immunosuppressants (Azathioprine ± 5‐ASA/corticosteroids) (n = 16) in order to compare the RQ values for each therapeutic group. All investigated genes were found upregulated in the inflamed mucosa of IBD patients in the following order: IL‐6 > FoxP3 > TGF&bgr;1 > IL‐23 > IL‐17A > IL‐10. We also observed that the gene expression of FoxP3 and IL‐6 were substantially higher in the inflamed mucosal tissue of the IBD patients than the adjacent normal mucosa (p = 0.035, p = 0.03 respectively). Differences between higher mRNA expression of FoxP3 and IL‐6 in inflamed tissue were considered significant in patients with ulcerative colitis (UC) (p = 0.011, p = 0.000 respectively) and with Crohns disease (CD) (p = 0.008, p = 0.000 respectively) in comparison to the normal mucosa of non‐IBD persons and we found increased TGF&bgr;1 in CD patients alone (p = 0.041). Furthermore, IL‐6 and TGF&bgr;1 were overexpressed (RQ > 10) in non‐inflamed mucosa from IBD patients compared to the normal mucosa from the controls. When we compared the gene expression for paired mucosa in the immunosuppressive treated group with the 5‐ASA treated group we observed opposite changes in IL‐6 and TGF&bgr;1 expression. Additionally, we found higher serum levels of IL‐23 (p = 0.008), TGF&bgr;1 and IL‐6 in IBD patients compared to non‐IBD patients. The obtained specific expression profile consisting of IL‐6, TGF&bgr;1, IL‐10 and FoxP3 may represent a transcriptional hallmark for IBD. Furthermore, we found that treatment with immunosuppressive therapy was more beneficial for driving cytokine expression to restore immune regulation in patients with IBD, unlike the 5‐ASA therapy.

Progesterone-modulated phosphatidylserine externalization in apoptosis and activation of Jurkat cells.

Problem  During pregnancy the elevated levels of progesterone (Pg) have immunomodulating effects. It is important to follow‐up Pg effects on basic biological processes at cell level as apoptosis and activation which was the aim of this study.