Generation of dendritic cells: A critical comparison of different methodological approaches

Dendritic cells (DCs) represent the most important antigen presenting cells (APCs). In vivo they originate from bone marrow myeloid and lymphoid precursors. In a first stage, the so-called immature DCs stay into nonlymphoid tissues and show high capacity of antigen capture and processing, but low T cell stimulatory capacity. After the internalization of the antigen, in the presence of inflammatory mediators, DCs mature and migrate out of nonlymphoid tissues into the blood or lymph, reaching secondary lymphoid organs. In this second stage the mature DCs lose the ability to capture antigens and increase the capacity to stimulate T cells, controlling the immune responses. Indeed, DCs are a family of heterogeneous cells and each subset exerts control over a different area of immunity, activating innate and acquired systems or maintaining the tolerance to antigens. For in vitro studies, dendritic cells can be isolated directly from peripheral blood through blood dendritic cell antibodies (BDCA), but they have been difficult to obtain due to their low concentration. So a wide variety of methods to have DCs has been performed, including the generation from circling CD14+ monocytes or from CD34+ progenitors. A multitude of systems have been carried out also to obtain these precursor cells. The method employed could affect morphological and functional features of the resultant DCs. For example, Ratta M and co-workers affirm that peripheral blood CD34+ cells generate a significantly higher number of fully functional DCs, capable of processing and presenting soluble antigens to autologous T cells. Elkord E and coworkers state that DCs generated from a positive immunoselected monocytes drastically reduce their ability to secrete cytokines as interleukin (IL)-12, IL-10, TNF-α, if compared to those generated from plastic adherence-isolated monocytes. Our own results indicate that monocytes selected by a positive selection method originate partially-mature DCs also without a maturational stimulus, characterized by low capacity of internalizing antigens and of inducing T cell proliferation also after lipopolysaccharide addition. Accordingly, in this commentary we review the recent progress in understanding the correlation between the approach used to obtain dendritic cells and their related properties, in order to indicate a correct application of DCs for research or clinical studies

The Centenary of Immune Thrombocytopenia - Part 1: Revising Nomenclature and Pathogenesis

The natural history of the immune thrombocytopenia (ITP) is interesting and intriguing because it traces different steps underlying autoimmune diseases. The review points out the main steps that have accompanied the stages of its history and the consequential changes related to its terminology. ITP is an autoimmune disease resulting from platelet antibody-mediated destruction and impaired megakaryocyte and platelet production. However, research advances highlight that a complex dysregulation of the immune system is involved in the pathogenesis of this condition. The review examines the role of the multiple immune components involved in the autoimmunity process, focusing on the more recent mechanisms, which could be new promising therapeutic targets for ITP patients

The effects of zoledronate on monocyte-derived dendritic cells from melanoma patients differ depending on the clinical stage of the disease.

Zoledronic acid has shown indirect anticancer effects on angiogenesis, the tumor microenvironment and immune responses. Its immunological action is exerted, at least in part, via its modulating properties. The aim of this study was to investigate the in vitro effects of zoledronic acid on the dendritic cells of melanoma patients. Peripheral blood samples were collected from 26 patients with melanoma and 11 healthy donors. Dendritic cells were derived from purified monocytes, and zoledronic acid (ZA) was added on the first day of culture. The phenotype and function of the generated cells were evaluated by flow cytometry. The ZA-treated monocytes from patients with early-stage disease generated DCs characterized by reduced endocytic activity and increased allostimulatory capacity compared with the untreated samples, allowing restoration of the DC function observed in normal subjects. In contrast, the ZA-treated monocytes from patients at stage III generated cells with higher CD14 antigen expression and endocytosis than the untreated samples. Therefore, in melanoma patients, the in vitro ZA effects differ according to the progression of the disease. In addition, our preliminary results appear to suggest that ZA effects are also influenced by the expression of CD14 antigen, indicating that the DC phenotype together with clinical characteristics must be considered in the choice of patients to be treated with ZA. Our work focus on the effect of ZA on monocyte-derived DCs from melanoma patients, showing that the effects of therapeutic doses of this drug might be mediated at least in part by modulation of myeloid cell function

Efficacy and Feasibility of the Epithelial Cell Adhesion Molecule (EpCAM) Immunomagnetic Cell Sorter for Studies of DNA Methylation in Colorectal Cancer

The aim of this work was to assess the impact on measurements of methylation of a panel of four cancer gene promoters of purifying tumor cells from colorectal tissue samples using the epithelial cell adhesion molecule (EpCAM)-immunomagnetic cell enrichment approach. We observed that, on average, methylation levels were higher in enriched cell fractions than in the whole tissue, but the difference was significant only for one out of four studied genes. In addition, there were strong correlations between methylation values for individual samples of whole tissue and the corresponding enriched cell fractions. Therefore, assays on whole tissue are likely to provide reliable estimates of tumor-specific methylation of cancer genes. However, tumor cell tissue separation using immunomagnetic beads could, in some cases, give a more accurate value of gene promoter methylation than the analysis of the whole cancer tissue, although relatively expensive and time-consuming. The efficacy and feasibility of the immunomagnetic cell sorting for methylation studies are discussed

Combined antiretroviral therapy reduces hyperimmunoglobulinemia in HIV-1 infected children

Objective: To evaluate the effect of combined antiretroviral therapy on serum immunoglobulin (Ig) levels in HIV-1 perinatally infected children. Methods: Data from 1250 children recorded by the Italian Register for HIV Infection in Children from 1985 to 2002 were analysed. Since Ig levels physiologically vary with age, differences at different age periods were evaluated as differences in z-scores calculated using means and standard deviations of normal population for each age period. Combined antiretroviral therapy has become widespread in Italy since 1996, thus differences in Ig z-scores between the periods 1985-1995 and 1996-2002 were analysed. Data according to type of therapeutic regimen were also analysed. Results: Between the two periods 1985-1995 and 1996-2002, significant (P < 0.0001) decreases in IgG (6.29 ± 4.72 versus 4.44 ± 4.33), IgM (9.25 ± 13.32 versus 5.61 ± 7.93), and IgA (10.25 ± 15.68 versus 6.48 ± 11.56) z-scores, together with a parallel significant (P < 0.0001) increase in CD4 T-lymphocyte percentages, were found. These decreases were confirmed regardless of whether the children were receiving intravenous Ig or not. Ig z-scores were significantly higher in children receiving mono-therapy than in those receiving double-combined therapy (IgC, P < 0.0001; IgM, P = 0.003; IgA, P = 0.031) and in the latter children than in those receiving three or more drugs (P < 0.0001 for all z-scores). Ig z-scores correlated inversely with CD4 T-lymphocyte percentages and, directly, with viral loads. Conclusions: Our data show that in HIV-1 infected children combined antiretroviral therapy leads to reduction of hyperimmunoglobulinemia which parallels restoration of CD4 T-lymphocyte percentage and viral load decrease, which it turn probably reflects improved B-lymphocyte functions. © 2004 Lippincott Williams & Wilkins

EFFETTI DI CITOCHINE SULLA CRESCITA DI PRECURSORI CELLULARI B UMANI PURIFICATI E IN PRESENZA DI COMPONENTE (CD13+) STROMALE

As reproducible models for examining human early B-cell progenitors (BCPs) are poorly developed, the cells and molecules regulating their growth and differentiation are still incompletely characterized. We used a recently published short term culture system, using immunomagnetic beads and negative selection, in order to isolate an early BCPs enriched population from human fetal tissues that support further studies on B cell proliferation or differentiation events. This purified population was incubated with or without human recombinant Interleukin-4 (rIL4), and its capability to proliferate and differentiate was followed. We found that rIL4 did not induce either proliferation or differentiation of purified human BCPs. Furthermore in the presence of stromal cells (CD13+) it was able to enhance cy mu + cells and to induce the expression of surface Ig (sIg), surface CD22 on in vitro TdT + CD19 + CD10 + sIg-fetal liver cells. Human recombinant interleukin-7 (rIL-7) promoted the proliferation and the clonal growth of Tdt + CD19 + CD10 + fetal BCPs, confirming its critical role at early stages of human B lymphopoiesis. Furthermore rIL7 also induced growth of CFU-GM when unseparated fetal tissues or myeloid/monocytic contaminated BCPs were used as a target populations, probably by indirect mechanism. Transforming growth factor -beta (TGF-beta 1) partially inhibited the stromal cell-dependent rIL4 induced differentiation and rIL7 clonal growth and proliferation of fetal BCPs. Our study contributes to elucidate the growth factor requirements that characterize normal human B-cell ontogeny, suggesting another mechanism for the linkage between lymphopoiesis and myeloid/macrophagic micro-environment. The in vivo implications of this study are discussed