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Biomedical subjects

S Ferrini

Publications and source records attributed to S Ferrini.

At least 19 recordsLinked to original sources
PubMed2006

Magnetic resonance imaging at 1.5 T with immunospecific contrast agent in vitro and in vivo in a xenotransplant model.

OBJECT: Demonstrating the feasibility of magnetic resonance imaging (MRI) at 1.5 T of ultrasmall particle iron oxide (USPIO)-antibody bound to tumor cells in vitro and in a murine xenotransplant model. METHODS: Human D430B cells or Raji Burkitt lymphoma cells were incubated in vitro with different amounts of commercially available USPIO-anti-CD20 antibodies and cell pellets were stratified in a test tube. For in vivo studies, D430B cells and Raji lymphoma cells were inoculated subcutaneously in immunodeficient mice. MRI at 1.5 T was performed with T1-weighted three-dimensional fast field echo sequences (17/4.6/13 degrees ) and T2-weighted three-dimensional fast-field echo sequences (50/12/7 degrees ). For in vivo studies MRI was performed before and 24 h after USPIO-anti-CD20 administration. RESULTS: USPIO-anti-CD20-treated D430B cells, showed a dose-dependent decrease in signal intensity (SI) on T2*-weighted images and SI enhancement on T1-weighted images in vitro. Raji cells showed lower SI changes, in accordance to the fivefold lower expression of CD20 on Raji with respect to D430B cells. In vivo 24 h after USPIO-anti-CD20 administration, both tumors showed an inhomogeneous decrease of SI on T2*-weighted images and SI enhancement on T1-weighted images. CONCLUSIONS: MRI at 1.5 T is able to detect USPIO-antibody conjugates targeting a tumor-associated antigen in vitro and in vivo.

Animals↗
PubMed2004

Low-dose interferon-gamma-producing human neuroblastoma cells show reduced proliferation and delayed tumorigenicity.

Interferon-gamma (IFN-gamma) directs T helper-1 cell differentiation and mediates antitumour effects in preclinical models. However, high-dose IFN-gamma is toxic in vivo, and IFN-gamma-transfected neuroblastoma (NB) cells secreting high amounts of the cytokine may be lost due to cell apoptosis or differentiation. Two human NB cell lines (ACN and SK-N-BE2(c)) differing as to genetic and phenotypic features were transfected with the human IFN-gamma gene and selected on the grounds of the low concentrations of IFN-gamma produced. In both IFN-gamma-transfected cell lines, autocrine and paracrine activation of IFN-gamma-mediated pathways occurred, leading to markedly reduced proliferation rate, to increased expression of surface HLA and CD40 molecules and of functional TNF binding sites. ACN/IFN-gamma cells showed a significantly delayed tumorigenicity in nude mice as compared to parental cells. ACN/IFN-gamma tumours were smaller, with extensive necrotic area as a result of a damaged and defective microvascular network. In addition, a significant reduction in the proliferation index was observed. This is the first demonstration that IFN-gamma inhibits in vivo proliferation of NB cell by acting on the tumour cell itself. This effect adds to the immunoregulatory and antiangiogenic activities operated by IFN-gamma in syngeneic tumour-bearing hosts.

Animals↗
PubMed2004

Prevention of angiogenesis by naked DNA IL-12 gene transfer: angioprevention by immunogene therapy.

IL-12 is thought to induce a cytokine cascade with antiangiogenic effects mediated by IFN-gamma and angiostatic CXCR3 chemokine ligands. Naked DNA intramuscular injection of an expression vector plasmid producing IL-12 resulted in significant, well-tolerated elevation of serum IL-12 levels. Injection of the IL-12 plasmid at least 2 days, and up to 20 days, before subcutaneous injection of matrigel with angiogenic factors resulted in strong prevention of angiogenesis in both C57/bl and nude mice. A single injection of the IL-12 plasmid contemporarily with the matrigel or 2 days after resulted in partial, statistically not significant, inhibition. Control plasmid injection did not affect either angiogenesis or angiogenesis inhibition by IL-12 protein in vivo. Angiogenesis inhibition was observed in NK cell-depleted C57/bl and nude mice as well as in IFN-gamma(-/-) and CXCR3(-/-) knockout mice, indicating that NK- and/or T-cell-initiated IFN-gamma-chemokine cascades were not involved in the angiogenesis inhibition observed in vivo. Finally, IL-12 plasmid DNA gene transfer significantly prevented the growth and vascularization of highly angiogenic KS-Imm Kaposi's sarcoma and TS/A murine mammary carcinoma tumors in nude and/or syngeneic mice. These data suggest that a preventive gene therapy approach using antiangiogenic cytokines can effectively inhibit tumor angiogenesis and KS, representing an example of angioimmunoprevention.

Animals↗
PubMed2003

Biological and clinical role of p73 in neuroblastoma.

The p73 gene is a p53 homologue localized at 1p36.3, a chromosomal region frequently deleted in neuroblastoma. p73 was originally considered an oncosuppressor gene. However, it was soon realized that its mode of action did not resemble that of a classic anti-oncogene. The recent discovery of N-terminal truncated isoforms, with oncogenic properties, showed that p73 has a 'two in one' structure. Indeed, the full-length variants are strong inducers of apoptosis while the truncated isoforms inhibit the pro-apoptotic activity of p53 and of the full-length p73. This review summarizes some aspects of p73 biology with particular reference to its possible role in neuroblastoma.

Alternative Splicing↗
PubMed2003

Detection of a functional hybrid receptor gammac/GM-CSFRbeta in human hematopoietic CD34+ cells.

A functional hybrid receptor associating the common gamma chain (gammac) with the granulocyte/macrophage colony-stimulating factor receptor beta (GM-CSFRbeta) chain is found in mobilized human peripheral blood (MPB) CD34+ hematopoietic progenitors, SCF/Flt3-L primed cord blood (CB) precursors (CBPr CD34+/CD56-), and CD34+ myeloid cell lines, but not in normal natural killer (NK) cells, the cytolytic NK-L cell line or nonhematopoietic cells. We demonstrated, using CD34+ TF1beta cells, which express an interleukin (IL)-15Ralpha/beta/gammac receptor, that within the hybrid receptor, the GM-CSFRbeta chain inhibits the IL-15-triggered gammac/JAK3-specific signaling controlling TF1beta cell proliferation. However, the gammac chain is part of a functional GM-CSFR, activating GM-CSF-dependent STAT5 nuclear translocation and the proliferation of TF1beta cells. The hybrid receptor is functional in normal hematopoietic progenitors in which both subunits control STAT5 activation. Finally, the parental TF1 cell line, which lacks the IL-15Rbeta chain, nevertheless expresses both a functional hybrid receptor that controls JAK3 phosphorylation and a novel IL-15alpha/gammac/TRAF2 complex that triggers nuclear factor kappaB activation. The lineage-dependent distribution and function of these receptors suggest that they are involved in hematopoiesis because they modify transduction pathways that play a major role in the differentiation of hematopoietic progenitors.

Antibodies, Monoclonal↗
PubMed2001

In vitro stimulation of human tonsillar subepithelial B cells: requirement for interaction with activated T cells.

Human tonsillar subepithelial B cells, which are a marginal zone-equivalent B cell subset, respond readily to T-independent type 2 antigens, but not to polyclonal B cell activators in vitro. In this study, subepithelial (SE) B cells were induced to proliferate and mature into plasma cells when co-cultured with activated T cells. The response of SE B cells was not observed when co-cultures were carried out in transwell chambers or in the presence of blocking anti-LFA-1 antibodies, demonstrating the need for a close T-B cell interaction. The presence of soluble CD40 also prevented the B cell response in vitro suggesting a pivotal role of CD40-CD40 ligand interactions. The data are discussed in terms of the T cell dependence of marginal zone (MZ) B cell response and the possible existence of various MZ B cell subsets.

B-Lymphocytes↗
PubMed2001

Lack of HLA-class I antigens in human neuroblastoma cells: analysis of its relationship to TAP and tapasin expression.

We studied the constitutive and the interferon (IFN)-gamma-induced expression of HLA class I antigen heavy chain, beta2-microglobulin (beta2m), TAP-1, TAP-2 and tapasin in a panel of eleven neuroblastoma cell lines. Surface expression of HLA class I antigens was low in eight out of eight neuroblastoma cell lines bearing MYC-N amplification and/or 1p deletion, while two out of three neuroblastoma cell lines lacking these genetic alterations showed normal expression. IFN-gamma treatment restored HLA class I antigen surface expression in all neuroblastoma cell lines. Eight out of 11 neuroblastoma cell lines did not express TAP-1 mRNA and three of them also lacked TAP-2 mRNA. beta2 m mRNA was barely detectable or absent in five neuroblastoma cell lines, while tapasin mRNA was always expressed. IFN-gamma upregulated the expression of HLA class I heavy chain, beta2 m, TAP-1, TAP-2 and tapasin, as detected at mRNA or protein level. Post-transcriptional events were involved in altered TAP-1 and beta2 m expression in one peculiar neuroblastoma cell line. These data indicate that multiple mechanisms play a role in the HLA class I antigen-deficient phenotype of human neuroblastoma.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Prevention of spontaneous neu-expressing mammary tumor development in mice transgenic for rat proto-neu by DNA vaccination.

The HER-2/neu proto-oncogene is overexpressed in 20-30% of human breast cancers and is associated with high recurrence risk. The oncogenic potential of HER-2/neu, together with its elevated expression in tumors, cell surface localization, and immunogenicity in some patients, make this oncoprotein an ideal target for immunotherapeutic approaches. To test the efficacy of immune-based strategies in eliciting an antitumor response, we used the N#202 transgenic mouse model engineered to overexpress the rat neu proto-oncogene under the control of the mouse mammary tumor virus promoter; females of this line develop spontaneous focal mammary tumors by 6 months of age. Transgenic mice immunized intramuscularly with a HER-2 cDNA ligated into the VR1012 (VICAL) expression vector under the control of the cytomegalovirus promoter developed significantly fewer spontaneous tumors as compared with mice injected with the empty vector (P < 0.0001) or not injected (P = 0.0006). However, this protection was observed only when immunization was started in 3-month-old but not in 6-month-old mice. These data suggest that the xenogeneic HER-2 DNA sequence can break immune tolerance to rat neu in transgenic N#202 mice and induce protective immunity that impairs the neu oncogene-driven progression of mammary carcinogenesis. The preventive effect achieved by our immunological approach appeared not to be based on anti-neu specific B and T cell immune attacks but was more possibly based on different mechanisms including aspecific and inflammatory immunological responses.

Age Factors↗
PubMed2000

Human immunodeficiency virus transactivator protein (Tat) stimulates chemotaxis, calcium mobilization, and activation of human polymorphonuclear leukocytes: implications for Tat-mediated pathogenesis.

The extracellular activities of the human immunodeficiency virus (HIV) transactivator protein (Tat) include induction of angiogenesis and stimulation of monocyte migration. Here it is shown that polymorphonuclear leukocytes (PMNL), mostly neutrophils, rapidly invade in response to Tat in vivo and initiate the formation of new vessels. In vitro, Tat was chemotactic for PMNL and induced calcium (Ca(2+)) mobilization. Tat proteins with inactivating substitutions in the arginine-glycine-aspartic acid or basic domain were still active in inducing PMNL migration, whereas Tat peptides mapped the migration and Ca(2+) mobilization activity to a cysteine-rich core domain, previously described as a Tat "chemokine-like" region (peptide CysL(24-51)). Tat and the CysL(24-51) peptide also induced PMNL superoxide production and the release of the angiogenic factors interleukin-8 and vascular endothelial growth factor from PMNL. CysL(24-51) did not induce endothelial cell migration but was angiogenic in vivo. These data indicate that the Tat activity on PMNL is mediated by its chemokine-like region and that PMNL recruitment by Tat is linked to angiogenesis.

Amino Acid Substitution↗
PubMed2000

IL-15/IL-15Ralpha intracellular trafficking in human melanoma cells and signal transduction through the IL-15Ralpha.

There are two IL-15 isoforms and eight isoforms for the IL-15Ralpha chain whose biological role is poorly understood. Here, we have analysed the intracellular trafficking of IL-15 and IL-15Ralpha and tried to shed some light on their function(s). In IL-15/GFP CHO transfectants both IL-15 isoforms show nuclear localization. Two melanoma cell lines (MELP and MELREO) spontaneously expressing the IL-15 isoforms, display different intracellular trafficking of the IL-15/IL-15Ralpha complex. In MELP cells only IL-15Ralpha is detected inside the nucleus, whereas IL-15 and IL-15Ralpha assemble at the cell surface and are internalized. Moreover, the transducing molecule TRAF2 co-immunoprecipitates with IL-15Ralpha and may be deflected to TNFRI using anti-IL-15 blocking mAbs and TNF-alpha. By contrast, MELREO cells display IL-15Ralpha and IL-15 nuclear localization but only a partial co-localization of these molecules on the cell surface. In these cells, TRAF2 is strongly associated with IL-15Ralpha and cannot be deflected by any treatment. Since TRAF2 activates the transcription factor NF-kappaB, IL-15 through IL-15Ralpha, could have a role in the control of this pathway. Indeed, anti-IL-15 MaB inhibit the constitutive nuclear localization of NFkappaB and the phosphorylation of its inhibitor Ikappa-Balpha. Thus, IL-15Ralpha controls NF-kappaB activation, however differences in the intracellular trafficking of the IL-15 and/or IL-15Ralpha suggest a different biological role for this complex in MELP versus MELREO cells.

Animals↗
PubMed2000

The combined action of IL-15 and IL-12 gene transfer can induce tumor cell rejection without T and NK cell involvement.

The cooperative antitumor effects of IL-12 and IL-15 gene transfer were studied in the N592 MHC class I-negative small cell lung cancer cell line xenotransplanted in nude mice. N592 cells engineered to secrete IL-15 displayed a significantly reduced tumor growth kinetics, and a slightly reduced tumor take rate, while N592 engineered with IL-12 displayed only minor changes in their growth in nude mice. However, N592 cells producing both cytokines were completely rejected, and produced a potent local bystander effect, inducing rejection of coinjected wild-type tumor cells. N592/IL-12/IL-15 cells were completely and promptly rejected also in NK-depleted nude mice, while in granulocyte-depleted animals a slight delay in the rejection process was observed. Immunohistochemical analyses of the N592/IL-12/IL-15 tumor area in intact nude mice revealed the presence of infiltrating macrophages, granulocytes, and NK cells, and expression of inducible NO synthase and of secondary cytokines such as IL-1beta, TNF-alpha, and IFN-gamma, and at higher levels GM-CSF, macrophage-inflammatory protein-2, and monocyte chemoattractant protein-1. In NK cell-depleted nude mice, numerous macrophages and granulocytes infiltrated the tumor, and a strong expression of macrophage-inflammatory protein-2 and inducible NO synthase was also observed. Finally, macrophages cocultured with N592/IL-12/IL-15 produced NO in vitro, and inhibited tumor cell growth, further suggesting their role as effector cells in this model.

Adjuvants, Immunologic↗
PubMed2000

Gene transfer of a secretable form of IL-15 in murine adenocarcinoma cells: effects on tumorigenicity, metastatic potential and immune response.

IL-15 is an immunostimulatory cytokine with IL-2-like activities. To exploit the potential role of IL-15 in cancer immuno-/gene therapy, we engineered murine TS/A cells with different IL-15 cDNA constructs. Significant IL-15 secretion was achieved only by the use of a modified cDNA encoding for an IL-15 pre-protein bearing the IgK light chain signal peptide. Different TS/A clones (TS/A IL-15 C6, C23, C29) producing 390 to 1,600 pg/ml biologically active IL-15 showed reduced tumorigenicity when implanted s.c. in syngeneic mice and significantly reduced metastatic potential by i.v. injection. Tumorigenicity of s.c. TS/A IL-15 was restored in animals depleted of CD8(+) lymphocytes or of natural killer cells and partially in CD4(+)-depleted mice. TS/A IL-15 cells displayed a significantly reduced growth rate by s.c. implant in nude mice. Also, >50% syngeneic animals rejecting TS/A IL-15 were resistant to a subsequent rechallenge with wild-type tumor (TS/Apc), indicating induction of protective immunity against TS/A tumor-associated antigens (TAAs). Cytolytic T lymphocyte (CTL) activity, specifically inhibited by anti-CD3 antibodies, was inducible in the splenocytes of TS/A IL-15-immunized animals by mixed lymphocyte/tumor culture (MLTC), and IFN-gamma was released in the supernatant of MLTC, mainly by CD8(+) cells. Immunohistochemistry of the TS/A IL-15 tumor area revealed the presence of an inflammatory infiltrate with predominant natural killer, macrophage, and granulocyte components and expression of IFN-gamma as a distinctive secondary cytokine. Use of TS/A IL-15 mitomycin-treated cells for therapeutic vaccination in experimental TS/A metastasis was effective in 60% of animals treated; these animals showed no metastatic tumor growth.

Adenocarcinoma↗
PubMed2000

Dissimilar anti-tumour reactions induced by tumour cells engineered with the interleukin-2 or interleukin-15 gene in nude mice.

Interleukin (IL)-15 shares immuno-stimulatory properties with IL-2 and is a potent inducer of natural killer (NK) cell function. The major histocompatibility complex (MHC) class I-negative human small cell lung cancer (SCLC) cell line N592, engineered to express a modified IL-15 cDNA (N592/IL-15), secreted biologically active IL-15 (300-500 pg/ml), capable of boosting T-cell proliferation and NK activity 'in vitro'. The effect of IL-15 gene transfer on natural immunity 'in vivo' was assessed by xenotransplants in nude mice and compared with that of the IL-2 gene. N592 cells engineered with IL-2 (N592/IL-2) were promptly rejected, while N592/IL-15 displayed a significant delay in tumour growth and a slightly reduced take rate. However, in NK-depleted nude mice, N592/IL-15 displayed the same growth kinetics as unmodified N592 cells, and N592/IL-2 grew with slightly reduced kinetics. An impressive reactive cell infiltration, consisting mainly of macrophages and granulocytes, was associated with N592/IL-2 tumour rejection, while a more evident recruitment of NK cells was found in N592/IL-15 tumours. In both N592 transfected tumours, we found expression of chemoattractant molecules, such as granulocyte macrophage-colony stimulating factor (GM-CSF) and monocyte chemoattractant protein (MCP)-1, while macrophage inflammatory protein (MIP)-2 was produced by endothelial cells only in N592/IL-2 tumours. In this tumour, very few and severely damaged microvessels were found, while microvessels were numerous in N592/IL-15 tumours. The potent recruitment of NK cells mediated by IL-15 gene transfer suggests its possible therapeutic use in tumours lacking MHC class I.

Animals↗
PubMed2000

Highly stable oligomerization forms of HIV-1 Tat detected by monoclonal antibodies and requirement of monomeric forms for the transactivating function on the HIV-1 LTR.

The use of newly generated murine monoclonal antibodies directed against distinct epitopes of a functionally active, chemically synthesized HIV-1 Tat protein has permitted the identification of several molecular forms including monomers, dimers and trimers. Dimers and trimers are particularly stable and resistant to strong reducing conditions. Through epitope mapping it has been possible to demonstrate that the major immunodominant epitope is contained within the basic region of the Tat protein and is lost after oligomerization of the molecule. In contrast, N-terminal, C-terminal and conformation-dependent epitopes are still accessible to mAb specific recognition after Tat oligomerization. Moreover, by using a quantitative HIV-LTR transactivation assay depending upon exogenous Tat, we could extrapolate the amount of functional Tat produced by cell lines stably transfected with the viral transactivator. More importantly, we could show that only the monomeric form of exogenous Tat is the relevant functional form acting in cells harbouring the HIV-1 LTR promoter.

Alkylation↗
PubMed2000

Interleukin-2 induces cell cycle perturbations leading to cell growth inhibition and death in malignant mesothelioma cells in vitro.

Previous report indicated that Interleukin-2 (IL-2) is able to inhibit the growth of IL-2-receptor-positive cancer cell lines without any involvement of the immune system, through IL-2-induced alterations of the cell cycle kinetics. In this study we provide evidence that IL-2 exerts anti-proliferative effect on three human malignant mesothelioma (MMe) cells in vitro, while no effects were observed on normal human mesothelial cell (HMC) primary cultures. The growth inhibitory effect of IL-2 on neoplastic cells appeared to depend on the baseline proliferative status of these cells. Indeed, in highly proliferating MMe cells, we observed a reduction of malignant cells in the S-phase of the cell cycle, with an accumulation in G0/G1, followed by apotosis for longer incubations or exposure to higher doses. On the contrary, in MMe cells proliferating at lower rate, IL-2 induces only a late cytotoxic effect, leading to apoptosis, without significantly affecting the cell cycle. IL-2Rbeta mRNA was detectable by RT-PCR in all MMe cells, IL-2Ralpha mRNA in one only out the three assayed and IL-2Rgamma mRNA in none. In addition, mRNA specific for the IL-2Rbeta-associated Jak-1 tyrosine kinase was expressed in all MMe cell lines, further suggesting that IL-2Rbeta may play a role in the observed effects. Very low, albeit detectable, levels of IL-2Rbeta chain appeared to be expressed at the cell surface of MMe cells by indirect immunofluorescence and FACS analyses. Finally, Ca(++) fluxes were rapidly induced when MMe cells were exposed to exogenous IL-2.

Apoptosis↗