Renato Vanacore

Immune Cell Imbalance in Major Depressive and Panic Disorders

We investigated subsets of peripheral immunologic cells in 12 drug-free patients affected by major depression according to DSM-III-R criteria, and who had recent evidence of somatic diseases. They were compared with 10 drug-free depressives, with 10 patients with panic disorder, and with 12 healthy volunteers, all without somatic disease. The immune subsets were measured by flow cytometry. The results showed that both groups of depressives had the same abnormalities in immune cells compared with the healthy volunteers or the panic disorder patients; in particular they presented a lower number of CD3+, CD8+ and HLA-DR+. The patients with panic attacks did not differ from healthy controls, except for CD4+ cells which were significantly lowered, even in comparison with the depressive groups. These data, although preliminary and in a small sample, suggest that some immune parameters may be influenced by the presence of a major psychiatric disorder.

Human Glioblastoma Multiforme: p53 Reactivation by a Novel MDM2 Inhibitor

Cancer development and chemo-resistance are often due to impaired functioning of the p53 tumor suppressor through genetic mutation or sequestration by other proteins. In glioblastoma multiforme (GBM), p53 availability is frequently reduced because it binds to the Murine Double Minute-2 (MDM2) oncoprotein, which accumulates at high concentrations in tumor cells. The use of MDM2 inhibitors that interfere with the binding of p53 and MDM2 has become a valid approach to inhibit cell growth in a number of cancers; however little is known about the efficacy of these inhibitors in GBM. We report that a new small-molecule inhibitor of MDM2 with a spirooxoindolepyrrolidine core structure, named ISA27, effectively reactivated p53 function and inhibited human GBM cell growth in vitro by inducing cell cycle arrest and apoptosis. In immunoincompetent BALB/c nude mice bearing a human GBM xenograft, the administration of ISA27 in vivo activated p53, inhibited cell proliferation and induced apoptosis in tumor tissue. Significantly, ISA27 was non-toxic in an in vitro normal human cell model and an in vivo mouse model. ISA27 administration in combination with temozolomide (TMZ) produced a synergistic inhibitory effect on GBM cell viability in vitro, suggesting the possibility of lowering the dose of TMZ used in the treatment of GBM. In conclusion, our data show that ISA27 releases the powerful antitumor capacities of p53 in GBM cells. The use of this MDM2 inhibitor could become a novel therapy for the treatment of GBM patients.

Lymphocyte subsets in multiple sclerosis A study with two-colour fluorescence analysis

Multiple sclerosis (MS) is postulated to be an immunopathologically mediated disease. This concept is supported by the finding of abnormally distributed peripheral blood T-cell subsets and a decreased T-suppressor function. Thirty-seven MS patients have been selected according to the criteria for definite MS. Fluorescein- or phycoerythrin-conjugated monoclonal antibodies have been used to define different lymphocyte subsets: CD4+, CD5+, CD8+, CD19+, CD38+, CD45RA+, CD4+CD45RA+, CD19+CD5+, CD8+CD38+. In relapsing-remitting (RR)-MS patients a significantly decreased percentage of CD19+ cells and in progressive MS patients a significantly increased percentage of CD19+CD5+ cells have been found. During a relapse in RR-MS, a significantly decreased percentage of CD4+CD45RA+ cells and a significantly increased percentage of CD8+CD38+ cells have been observed. Moreover, in RR-MS patients a significantly increased percentage of CD38+ cells and significantly high IgM amounts have been found. The increased percentage of CD19+CD5+ and CD38+ cells (together with high IgM levels) and the reduced percentage of CD4+CD45RA+ lymphocytes could be related to an activation of both cellular and humoral immune response in acute MS.

PK 11195 differentially affects cell survival in human wild-type and 18 kDa translocator protein-silenced ADF astrocytoma cells†

Gliomas are the most common brain tumours with a poor prognosis due to their aggressiveness and propensity for recurrence. The 18 kDa translocator protein (TSPO) has been demonstrated to be greatly expressed in glioma cells and its over‐expression has been correlated with glioma malignance grades. Due to both its high density in tumours and the pro‐apoptotic activity of its ligands, TSPO has been suggested as a promising target in gliomas. With the aim to evidence if the TSPO expression level alters glioma cell susceptibility to undergo to cell death, we analysed the effects of the specific TSPO ligand, PK 11195, in human astrocytoma wild‐type and TSPO‐silenced cell lines. As first step, TSPO was characterised in human astrocytoma cell line (ADF). Our data demonstrated the presence of a single class of TSPO binding sites highly expressed in mitochondria. PK 11195 cell treatment activated an autophagic pathway followed by apoptosis mediated by the modulation of the mitochondrial permeability transition. In TSPO‐silenced cells, produced by siRNA technique, a reduced cell proliferation rate and a decreased cell susceptibility to the PK 11195‐induced anti‐proliferative effect and mitochondrial potential dissipation were demonstrated respect to control cells. In conclusion, for the first time, PK 11195 was demonstrated to differentially affect glioma cell survival in relation to TSPO expression levels. These results encourage the development of specific‐cell strategies for the treatment of gliomas, in which TSPO is highly expressed respect to normal cells. J. Cell. Biochem. 105: 712–723, 2008.

Different media and supplements modulate the clonogenic and expansion properties of rabbit bone marrow mesenchymal stem cells

Background -Rabbits provide an excellent model for many animal and human diseases, such as cardiovascular diseases, for the development of new vaccines in wound healing management and in the field of tissue engineering of tendon, cartilage, bone and skin.The study presented herein aims to investigate the biological properties of bone marrow rabbit MSCs cultured in different conditions, in order to provide a basis for their clinical applications in veterinary medicine.Findings -MSCs were isolated from 5 New Zealand rabbits. Fold increase, CFU number, doubling time, differentiation ability and immunophenotype were analyzed.With the plating density of 10 cells/cm2 the fold increase was significantly lower with DMEM-20%FCS and MSCs growth was significantly higher with αMEM-hEGF. The highest clonogenic ability was found at 100 cell/cm2 with MSCBM and at 10 cell/cm2 with M199. Both at 10 and 100 cells/cm2, in αMEM medium, the highest CFU increase was obtained by adding bFGF. Supplementing culture media with 10%FCS-10%HS determined a significant increase of CFU.Conclusion -Our data suggest that different progenitor cells with differential sensitivity to media, sera and growth factors exist and the choice of culture conditions has to be carefully considered for MSC management.

Different growth conditions for peripheral blood endothelial progenitors

PURPOSE To compare different growth conditions for endothelial progenitor cells (EPCs) from peripheral blood mononuclear cells (PBMNCs). METHODS AND MATERIALS PBMNCs of healthy volunteers were cultured on fibronectin as follows: M199 with VEGF, bFGF, IGF-I; the same medium with bovine retina-derived extract (RDE); freshly isolated or depleted of adherent cells PBMNCs in HUVEC conditioned medium; DiI-stained PBMNCs with HUVECs (1:4 ratio) in Ml99 with RDE. PBMNCs were analysed by FACS using mAbs for endothelial markers. EPCs migration was determined using a modified Boyden chamber assay and VEGF as chemoattractant. EPCs were seeded alone or with HUVECs on Matrigel to assess in vitro angiogenesis. RESULTS With growth factors, numerous cell clusters appeared within 1 week. Spindle-shaped and attached cells sprouted, differentiating in endothelial cell (EC)-like cells within 2 weeks and forming cobblestone-like monolayers within 3 weeks. With RDE, numerous large cell clusters appeared within 1 week, but the number of cells with an EC morphology decreased during culture. FACS confirmed the endothelial phenotype and attached cells were able to migrate in response to VEGF. When nonadherent cells were cultured in HUVEC conditioned medium, they proliferated readily and EPCs were induced while freshly isolated cells neither proliferated nor induced EPCs. FACS analysis of the cocultures showed the presence of double-labeled PBMNCs expressing endothelial antigens. Capillary-like structures were observed on Matrigel only from cocultures and PBMNCs were able to incorporate in these networks. CONCLUSIONS PBMNCs are able to differentiate in EPCs when stimulated with appropriate culture conditions (growth factors, HUVEC conditioned medium, HUVECs).

FcRIII (CD16) expression on neutrophils from chronic myeloid leukemia. A flow cytometric study

FcRIII (CD16) expression on neutrophils from 17 patients with chronic myeloid leukemia (CML) was studied by flow cytometry using monoclonal antibodies. A variable proportion of CD16-negative neutrophils were found both in CML patients in chronic phase (3 out of 8 patients) and in CML patients in hematological remission (3 out of 9 patients). Neutrophils with reduced FcRIII expression showed more defective chemiluminescence and phagocytosis than neutrophils with normal FcRIII expression. Circulating myeloid cells from three patients in chronic phase, showing a normal percentage of CD16-positive neutrophils, were isolated and fractionated by discontinuous Percoll gradients. This study showed that CD16 appears at the stage of metamyelocyte, that band cells and segmented neutrophils display an identical pattern of membrane FcRIII, and that the fluorescence intensity shown by metamyelocytes is different from that displayed by more mature cells. The association between low FcRIII expression and function abnormality could be suggestive of a defect in CML neutrophil maturation.

The mechanisms of nadroparin-mediated inhibition of proliferation of two human lung cancer cell lines.

Clinical data suggest that heparin treatment improves survival of lung cancer patients, but the mechanisms involved are not fully understood. We investigated whether low molecular weight heparin nadroparin, directly affects lung cancer cell population growth in conventionally cultured cell lines.

Pre-birth selection of umbilical cord blood donors.

BACKGROUND . The fact that only a small percentage of cord blood units (CBU) stored are actually used for transplantation contributes to raising the already high costs of their processing and cryopreservation. The identification of predictors allowing the early identification of suitable CBU would allow a reduction of costs for the collection, storage and characterisation of CBU with insufficient volume or cell numbers. In our bank we have adopted a cut-off value for using CBU of 8 x 10(8) nucleated cells and a volume >or= 60 mL. MATERIALS AND METHODS In 365 banked CBU, we evaluated the correlation between neonatal/gestational parameters and laboratory data used to assess their quality. RESULTS Biparietal diameter (BPD) and abdominal circumference were significantly and positively correlated with CBU volume (r(2)=0.12, p=0.0011 and r(2)=0.092, p=0.0063, respectively). Receiver operating characteristic (ROC) analysis showed that both parameters can be used to identify CBU with insufficient volume (BPD: area under the curve 0.69, 95% CI=0.57-0.82, p=0.004; abdominal circumference: area under the curve 0.67, 95% CI=0.54-0.79, p<0.01). BPD and head circumference, but not abdominal circumference or femoral length, were positively correlated with white blood cell (WBC) count (r(2)=0.215, p=0.031, and r(2)=0.299, p=0.015, respectively). Abdominal circumference, but not BPD, head circumference or femoral length, was statistically significantly correlated with the number of CD34(+) cells in the CBU. Weight at birth and placental weight were positively correlated with WBC count, blood volume, CD34(+) cell count, total colony-forming units and burst-forming units. CONCLUSION . Pre-birth assessment of BPD might allow the selection of donors who would yield CBU of sufficient volume and WBC count and avoid the costs of collecting, transferring, storing and analysing CBU with a high probability of resulting unsuitable for transplantation.

PIGA (N,N-Di-n-butyl-5-chloro-2-(4-chlorophenyl)indol-3-ylglyoxylamide), a new mitochondrial benzodiazepine-receptor ligand, induces apoptosis in C6 glioma cells

Mitochondrial benzodiazepine‐receptor (mBzR) ligands constitute a heterogeneous class of compounds that show a pleiotropic spectrum of effects within the cells, including the modulation of apoptosis. In this paper, a novel synthetic 2‐phenylindol‐3‐ylglyoxylamide derivative, N,N‐di‐n‐butyl‐5‐chloro‐2‐(4‐chlorophenyl)indol‐3‐ylglyoxylamide (PIGA), which shows high affinity and selectivity for the mBzR, is demonstrated to induce apoptosis in rat C6 glioma cells. PIGA was able to dissipate mitochondrial transmembrane potential (ΔΨm) and to cause a significant cytosolic accumulation of cytochrome c. Moreover, typical features of apoptotic cell death, such as caspase‐3 activation and DNA fragmentation, were also detected in PIGA‐treated cells. Our data expand the knowledge on mBzR ligand‐mediated apoptosis and suggest PIGA as a novel proapoptotic compound with therapeutic potential against glial tumours, in which apoptosis resistance has been reported to be involved in carcinogenesis.