Annalisa Legitimo

Morpho-functional characterization of human mesenchymal stem cells from umbilical cord blood for potential uses in regenerative medicine

Mesenchymal stem cells (MSCs) represent a promising source of progenitor cells having the potential to repair and to regenerate diseased or damaged skeletal tissues. Bone marrow (BM) has been the first source reported to contain MSCs. However, BM-derived cells are not always acceptable, due to the highly invasive drawing and the decline in MSC number and differentiative capability with increasing age. Human umbilical cord blood (UCB), obtainable by donation with a noninvasive method, has been introduced as an alternative source of MSCs. Here human UCB-derived MSCs isolation and morpho-functional characterization are reported. Human UCB-derived mononuclear cells, obtained by negative immunoselection, exhibited either an osteoclast-like or a mesenchymal-like phenotype. However, we were able to obtain homogeneous populations of MSCs that displayed a fibroblast-like morphology, expressed mesenchym-related antigens and showed differentiative capacities along osteoblastic and early chondroblastic lineages. Furthermore, this study is one among a few papers investigating human UCB-derived MSC growth and differentiation on three-dimensional scaffolds focusing on their potential applications in regenerative medicine and tissue engineering. UCB-derived MSCs were proved to grow on biodegradable microfiber meshes; additionally, they were able to differentiate toward mature osteoblasts when cultured inside human plasma clots, suggesting their potential application in orthopedic surgery.

Enumeration of human peripheral blood dendritic cells throughout the life

Human aging is associated with immunosenescence, a process characterized by alterations in numerical and functional features of immune system components. Dendritic cells (DCs) are the main antigen-presenting cells, playing a pivotal role in adaptive and innate immunity. Therefore, we investigated the distribution of human circulating DCs throughout the life, in order to contribute to the knowledge of the physiological background underlying the aging of immune system. Cytofluorimetric analysis of peripheral blood samples by all-aged healthy population showed a significant decrease of circulating DCs and of their two main subsets among age. This reduction was limited to the plasmacytoid cell subtype when young and old subjects were analyzed separately. The analysis of circulating Treg cell number in a cohort of the subjects showed a significant reduction with increasing age and a positive significant correlation to myeloid or plasmacytoid absolute numbers. In conclusion, this work provides a large set of data of normal reference values of peripheral blood dendritic cells in healthy population suitable for comparative clinical studies concerning pathological immune dysfunctions.

Gene promoter methylation in colorectal cancer and healthy adjacent mucosa specimens: Correlation with physiological and pathological characteristics, and with biomarkers of one-carbon metabolism

We evaluated the promoter methylation levels of the APC, MGMT, hMLH1, RASSF1A and CDKN2A genes in 107 colorectal cancer (CRC) samples and 80 healthy adjacent tissues. We searched for correlation with both physical and pathological features, polymorphisms of folate metabolism pathway genes (MTHFR, MTRR, MTR, RFC1, TYMS, and DNMT3B), and data on circulating folate, vitamin B12 and homocysteine, which were available in a subgroup of the CRC patients. An increased number of methylated samples were found in CRC respect to adjacent healthy tissues, with the exception of APC, which was also frequently methylated in healthy colonic mucosa. Statistically significant associations were found between RASSF1A promoter methylation and tumor stage, and between hMLH1 promoter methylation and tumor location. Increasing age positively correlated with both hMLH1 and MGMT methylation levels in CRC tissues, and with APC methylation levels in the adjacent healthy mucosa. Concerning gender, females showed higher hMLH1 promoter methylation levels with respect to males. In CRC samples, the MTR 2756AG genotype correlated with higher methylation levels of RASSF1A, and the TYMS 1494 6bp ins/del polymorphism correlated with the methylation levels of both APC and hMLH1. In adjacent healthy tissues, MTR 2756AG and TYMS 1494 6bp del/del genotypes correlated with APC and MGMT promoter methylation, respectively. Low folate levels were associated with hMLH1 hypermethylation. Present results support the hypothesis that DNA methylation in CRC depends from both physiological and environmental factors, with one-carbon metabolism largely involved in this process.

In vitro treatment of monocytes with 8-methoxypsolaren and ultraviolet A light induces dendritic cells with a tolerogenic phenotype

Extracorporeal photopheresis (ECP) has been considered an efficient dendritic cell (DC) therapy, used for treating both T cell malignancy, as well as T cell‐mediated diseases. During the ECP procedure leucocytes are exposed to photoactivable agent 8‐methoxypsolaren (8‐MOP) and ultraviolet (UV) A radiation (PUVA) prior to reinfusion. Despite its clinical efficacy the mechanism of action remains elusive. As it has been reported that ECP might promote the differentiation of monocytes into immature DCs, we investigated the effects of UVA light (2 J/cm2) and 8‐MOP (100 ng/ml) on in vitro monocyte‐to‐DC differentiation from normal donors. DCs were generated from human purified CD14+ cells. Because monocytes are killed by PUVA and taking into account that only 5–10% of circulating mononuclear cells are exposed to PUVA during the ECP procedure, we developed an assay in which 10% of PUVA‐treated monocytes were co‐cultured with untreated monocytes. We first demonstrate that the presence of 10% apoptotic cells and monocyte activation were not enough to induce monocyte differentiation into DCs. Adding cytokines to our culture system, we obtained immature DCs characterized by significantly higher phagocytic activity and human leucocyte antigen D‐related (HLA‐DR) expression. These DCs preserved the capacity to be activated by lipopolysaccharide, but showed a reduced capacity to induce allogeneic T cell proliferation when first co‐cultured with 10% of PUVA‐treated cells. Our experimental design provides a novel insight into the real action of 8‐MOP and UVA light on dendritic cell biology, suggesting an additional mechanism by which 8‐MOP and UVA light exposure may influence immune responses.

Immunological Evaluation of Patients with Beta-Thalassemia major

Abnormalities in the immune system and zinc homeostasis in patients with β-thalassemia major (TM) have been reported. Since zinc ion is essential for the efficiency of the immune system and is required to induce biological activity to thymulin (Zn-FTS), a biochemically defined thymic hormone, we investigated the plasma levels of zinc and both active thymulin (Zn-FTS) and total zinc saturable thymulin (Zn-FTS+FTS) in 18 patients with TM aged between 2 and 31 years and 22 normal controls of the same age. Inhibitory molecules anti-thymulin and the distribution of lymphocyte subsets were also analyzed. Patients with TM presented significantly lowered plasma zinc and thymulin levels when compared to normal subjects. The significant enhancement of the active form of the hormone after zinc addition in vitro suggests that low thymulin values found in TM are due not to a thymic failure in synthesizing and secreting the thymic hormone, but a defect in zinc saturation of the hormone. An impairment of cell subset distribution was also demonstrated. This study shows that zinc and thymulin deficiency contribute to the complex mechanisms underlying immune dysfunction in TM.

The methodological approach for the generation of human dendritic cells from monocytes affects the maturation state of the resultant dendritic cells

Dendritic cells (DCs) are effective as antigen-presenting cells in the immune system and are present at two functional stages depending on their maturation state. For experimental investigation of this concept, CD14(+) monocytes from blood are isolated and cultured to generate in vitro the DCs needed for functional analysis. For positive selection of CD14(+) monocytes we compared two immunomagnetic bead technologies: MACS Separation, created by Miltenyi Biotec, and EasySep Selection, created by StemCell Technologies. The monocytes provided dendritic cells for their functional analysis. Lipopolysaccharide was added to cultured DCs to induce maturation. Although both systems generated DCs from the positively selected CD14(+) cells, there were certain differences between them. Morphological, phenotypic, and functional analysis showed that MACS-selection provided DCs that have typical features corresponding to day 6 or 7 of maturation. EasySep-DCs exist in a partially-mature state from day 6 onward, even without the addition of a maturation stimulus. The reason behind this partial maturation is possibly based on the dextran-coated beads that are associated with the EasySep product. Both methods provide pure and viable DCs, but we would recommend using the MACS system for obtaining DCs suitable for functional studies.

Effect of laser micromachining of titanium on viability and responsiveness of osteoblast-like cells.

Objectives:Laser engineering may create hemispherical porosities on titanium surfaces obtaining regular and predetermined rough titanium surfaces. The aim of this study was to assess the viability and the proliferation of primary osteoblast-like cells (OB) to growth factors on titanium surfaces with a different roughness in vitro. Materials and Methods:OB were obtained from volunteers undergoing wisdom tooth removal following a standardized protocol. OB were allowed to attach on 4 different titanium surfaces: sandblasted titanium (SBT) disks, 5-, 10-, and 20-&mgr;m regular laser-engineered micropore titanium disks. A well with no disk was used as control. Cell morphology was evaluated with scanning electron microscopy. Viability was measured with MTT (3[4,5 dimethylthiazol 2yl]2,5 diphenyltetrazolium bromide) assay. Proliferation rate of attached cells was evaluated with Cell Counting Kit-8 48 hours after platelet-released supernatant (PRS) application. Statistical analysiswas performed with analysis of variance test. Results:All surfaces showed OB attachment on scanning electron microscopy. OB appeared more numerous on 20T surfaces. Laser-engineered surfaces showed higher OB viability than SBT (P < 0.01). In terms of proliferation, viability increase was noted for all groups after platelet-released supernatant application. 20T and SBT disks seemed to trigger the higher cellular proliferation (20T vs 10T, P < 0.05). Conclusions:Laser-engineered porous titanium surfaces promote viability and proliferation of OB. In particular, hemispherical porosity of 20 &mgr;m seems to trigger the higher OB response. Further research is needed to confirm these data.

Effects of vitamin D on the growth of normal and malignant B-cell progenitors

As the effects of vitamin D3, 1,25‐dihydroxyvitamin D3 (1,25‐(OH)2‐D3) (VD, calcitriol) on the proliferation and differentiation potential of normal and leukaemic cells in vitro of myeloid lineage are known, we investigated the response to VD on the growth of both normal and malignant lymphoid progenitors. Effects of vitamin D on normal human lymphoid progenitors and B lineage acute lymphoblastic leukaemia (ALL) progenitors were assessed by using an in vitro cell colony assay specific for either B or T cell lineages. The expression of VDR on B untreated malignant progenitors at diagnosis was investigated by RT‐PCR analysis. VD induced a significant inhibition of normal lymphoid cell progenitors growth of both T and B lineage. VD inhibited significantly also the growth of malignant B cell lineage lymphoid progenitors, without inducing cytotoxic effect. As it has been reported that VD effects on activated lymphocytes are mediated by 1,25‐(OH)2‐D3 nuclear receptor (VDR), we investigated VDR expression on malignant B cell progenitors. We did not detect VDR expression on these cells examined at diagnosis. We demonstrated that VD inhibited in vitro the clonogenic growth of both normal and malignant lymphoid B cell progenitors and that this inhibitory effect on malignant B cell progenitors was not related to VDR. Our work contributes to understanding of the mechanism of action of this hormone in promoting cellular inhibition of clonal growth of malignant lymphoid B cell progenitors, suggesting that the regulation of some critical growth and differentiation factor receptors could be a key physiological role of this hormone.

Defective Generation and Maturation of Dendritic Cells from Monocytes in Colorectal Cancer Patients during the Course of Disease

Colorectal cancer (CRC) is the second-leading cause of cancer-related deaths in Western countries. Today, the role of the host’s immune system in controlling the progression and spread of solid tumors is broadly established. Tumor immunosurveillance escape mechanisms, such as those involving dendritic cells (DCs), the most important antigen-presenting cells, are likewise recognized processes involved in cancer. The present study evaluates the ability of CRC patients to generate DCs in vitro from circulating monocytes at both pre- and post-operative timepoints; the results are correlated with the stage of disease to shed light on the systemic immune statuses of CRC patients. Our data showed that patients’ DCs had lower co-stimulatory molecule expression and were less able to present antigens to allogeneic T cells compared to healthy controls’ (HC) DCs. Furthermore altered cytokine secretion, such as increased IL-10 and reduced IL-12 and TNF-α, was observed. At the post-operative timepoints we observed a recovery of the patients’ ability to generate immature DCs, compared to HCs, but the maturational capacity remained affected. Our study conclusively highlights the persistently impaired in vitro generation of fully mature and functional DCs, which appears to be more altered during advanced stages. This work sheds light on a dendritic cell-based tumor immune escape mechanism that could be useful for the development of more effective immunotherapeutic strategies.

The challenge of culturing human colorectal tumor cells: establishment of a cell culture model by the comparison of different methodological approaches

Background Because colorectal cancer is a significant cause of morbidity and mortality in the Western population, knowledge of the molecular and biological alterations associated with its development is important. Since primary human colon cancer cultures from fresh tumor tissue are technically difficult to obtain, experiments in most laboratories are performed on colon epithelial cell lines, but these represent just one stage of tumor progression. Only primary cultures of neoplastic colonocytes may reflect the actual responsiveness of tumors at certain developmental stages to antitumor agents. Methods This paper analyzes several critical points concerning primary cultures, ranging from cell isolation to culture conditions, and compares different methodological approaches to isolate and cultivate a pure fraction of viable tumor cells. Samples of resected colorectal cancers were collected from 20 patients (stage T3 or T4). We compared in vitro several approaches of tissue disaggregation including mechanical disaggregation and enzymatic dissociation with trypsin or collagenase. Isolated cells were maintained in a short-term serum-free culture system. Evaluation of the purity and tumoral nature of isolated cells was performed by immunochemistry. Results We established the antibiotic concentration necessary during transport and washing of the specimens to prevent microbial overgrowth. We demonstrated that the number of viable cells was dependent on the dissociation method used. Mechanical disaggregation is not a valid dissociation method because of the high mortality of cells and might be used only in samples for molecular analysis. Comparison of the enzymatic digestion procedures showed that digestion with trypsin allowed the highest recovery of viable cells. Conclusion In this paper we analyzed several critical aspects of cell culture procedures and designed a methodological approach suitable for functional studies of colorectal cancer.