Biomedical subjects

F Belardelli

Publications and source records attributed to F Belardelli.

At least 55 records · Page 3Linked to original sources

PubMed1998

Antiviral effect of bovine lactoferrin saturated with metal ions on early steps of human immunodeficiency virus type 1 infection.

Lactoferrin is a mammalian iron-binding glycoprotein present in many biological secretions, such as milk, tears, semen and plasma and a major component of the specific granules of polymorphonuclear leucocytes. The effect of bovine lactoferrin (BLf) in apo-form or saturated with ferric, manganese or zinc ions, on human immunodeficiency virus type 1 (HIV-1) infection in the C8166 T-cell line was studied. Both HIV-1 replication and syncytium formation were efficiently inhibited, in a dose-dependent manner, by lactoferrins. BLf in apo and saturated forms markedly inhibited HIV-1 replication when added prior to HIV infection or during the virus adsorption step, thus suggesting a mechanism of action on the HIV binding to or entry into C8166 cells. Likewise, the addition of Fe3+BLf prior to HIV infection and during the attachment step resulted in a marked reduction of the HIV-1 DNA in C8166 cells 20 h after infection. The potent antiviral effect and the high selectivity index exhibited by BLf suggest for this protein, in apo or saturated forms, an important role in inhibiting the early HIV-cell interaction, even though a post adsorption effect cannot be ruled out.

Adsorption↗

PubMed1998

IFN-gamma expression in macrophages and its possible biological significance.

IFN-gamma is a pleiotropic cytokine endowed with potent immunomodulatory effects whose expression was long considered to be restricted to T and NK cells. Only recently, it became evident that IFN-gamma production can also occur in other cell types, including monocyte/macrophages. However, the biological relevance of macrophage IFN-gamma is still unclear. The purpose of this article is to provide an overview of the collected evidence demonstrating IFN-gamma expression in macrophages and to discuss the possible biological significance of this cytokine production in the early phase of host response to infectious agents.

Animals↗

PubMed1998

Inhibition of lung colonisation of a mouse mammary carcinoma by therapeutic vaccination with interferon-alpha gene-transduced tumor cells.

A spontaneously metastatic murine mammary adenocarcinoma, TSA, has been transduced with the gene for interferon alpha1 (IFN-alpha). Transfectants were used for the immunotherapy of mice bearing lung colonies induced by the intravenous inoculation of non-transduced parental cells. A significant reduction in the number of tumor colonies was obtained when repeated subcutaneous administrations of mitomycin C-blocked transfectant cells were given, commencing 3 days after an intravenous challenge with TSA cells. Intraperitoneal vaccination induced a stronger anti-tumor response than subcutaneous vaccination, and the proportion of tumor-free mice reached 50%. The potency of IFN-alpha transfectants was similar to that of IFN-gamma transfectants previously obtained from TSA. Admixture of IFN-alpha and IFN-gamma transfectant cells in the same vaccine did not increase the curative effect over that of single vaccines. In nude mice vaccination with IFN-alpha or IFN-gamma transfectants did not lead to a reduction in the number of lung colonies, indicating that an intact T cell response was required for the therapeutic effect observed in immunocompetent mice.

Adenocarcinoma↗

PubMed1998

Role of cytokines in GVL (ESb lymphoma) and GVHD after adoptive transfer of allogeneic T lymphocytes in mice.

ESb lymphoma cells injected i.v. into DBA/2 (H-2d) mice multiply rapidly in the liver and kill all mice in a few days. Adoptive transfer of allogeneic C57B1/6 (H-2b) tumor-immune or normal splenic lymphocytes to sublethally irradiated DBA/2 mice induced a marked antitumor state, graft-versus-leukemia (GVL), increasing the mean survival time 2-3-fold, but also induced an acute and lethal graft-versus host disease (GVHD). We have undertaken experiments to try to dissociate GVL from GVHD. Transfer of immune spleen cells induced a greater GVL than transfer of normal spleen cells with an equivalent to GVHD. Three to five million immune or normal CD8+ T lymphocytes were sufficient to induce both GVL and GVHD. Individual DBA/2 mice were labeled and followed. In mice undergoing GVHD, the spleens were repopulated by donor (H-2b) lymphocytes, and tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) were present in the sera of 26 of 27 and 18 of 20 mice, respectively, together with increased amounts of TNF-alpha and IL-6 mRNA in their spleens. This was in contrast to DBA/2 mice receiving allogeneic cells but not developing GVHD. Both interferon-alpha/beta (IFN-alpha/beta) and IL-12, which had proven very effective in association with adoptive transfer of syngeneic immune T lymphocytes in inhibiting ESb metastases, enhanced GVHD when administered with allogeneic immune or normal spleen cells, and >90% of mice died. Intensive IL-2 treatment inhibited GVHD while maintaining GVL.

Adoptive Transfer↗

PubMed1998

Human lymphoblastoid CD4(+) T cells become permissive to macrophage-tropic strains of human immunodeficiency virus type 1 after passage into severe combined immunodeficient mice through in vivo upregulation of CCR5: in vivo dynamics of CD4(+) T-cell differentiation in pathogenesis of AIDS.

In this article, we show that passage in SCID mice rendered a human CD4(+) T-cell line (CEM cells) highly susceptible to infection by macrophage-tropic (M-tropic) strains and primary clinical isolates of human immunodeficiency virus type 1 (HIV-1). This in vivo-acquired permissiveness of CEM cells was associated with the induction of a CD45RO+ phenotype as well as of some beta-chemokine receptors. Regulated upon activation, normal T-cell expressed and secreted chemokine entirely inhibited the ability of M-tropic HIV-1 strains to infect these cells. These findings may lead to new approaches in investigating in vivo the capacity of different HIV strains to exploit chemokine receptors in relation to the dynamics of the activation and/or differentiation state of human CD4(+) T cells.

Acquired Immunodeficiency Syndrome↗

PubMed1997

IL-12 induces IFN-gamma expression and secretion in mouse peritoneal macrophages.

We previously reported that resting mouse peritoneal macrophages (PM) constitutively express low levels of IFN-gamma, whose production is consistently enhanced by exogenous IFN-gamma. In this study, we investigated the effects of IL-12 on the replication of vesicular stomatitis virus and on IFN-gamma gene expression in mouse PM. The addition of IL-12 to freshly explanted PM resulted in the persistence of an antiviral state to vesicular stomatitis virus, while control PM progressively became permissive for virus replication after 3 to 4 days in culture. The IL-12-induced antiviral state was inhibited by Abs to IFN-gamma, suggesting that endogenous IFN-gamma was largely responsible for this antiviral response. Moreover, IL-12 induced a consistent secretion of IFN-gamma, especially in cultured PM. The IL-1 2-induced antiviral state and IFN-gamma production were observed using PM from various strains of mice, including LPS-defective C3H/HeJ, NK-deficient bg/bg, DBA/2, Swiss (CD1), and Swiss nude mice treated or not with anti-asialo GM1 Abs. A 4-h treatment with IL-12 was sufficient to induce a marked accumulation of IFN-gamma mRNA, which was greater in cultured PM than in freshly harvested cells. Lastly, immunofluorescence studies in IL-12-stimulated macrophages clearly showed an enhancement of immunoreactive IFN-gamma compared with basal levels in cells exhibiting a macrophage (i.e., F4/80-positive) phenotype. Together, these findings demonstrate that IL-12 can directly stimulate mouse PM to produce IFN-gamma. We suggest that IL-12-induced IFN-gamma production by macrophages can play some role in the generation of the antiviral and immunoregulatory effects of IL-12.

Animals↗

PubMed1997

Extracellular matrix remodelling in a murine mammary adenocarcinoma transfected with the interferon-alpha 1 gene.

The rejection of interferon alpha 1 gene-transfected mammary adenocarcinoma cells (TSA-IFN alpha) injected into syngeneic BALB/c mice was accompanied by an unusual stromal reaction and marked CD8-positive T-lymphocyte involvement. To investigate the biological background of this reaction, the possibility was evaluated that an interaction between TSA-IFN alpha and stromal cells might remodel the extracellular matrix (EM). When fibroblasts were co-cultured with TSA-IFN alpha or treated with exogenous IFN alpha, there was no change in their replication rate or collagen synthesis. By contrast, their fibronectin (FN) production and release were increased, resulting in enhanced fibroblast chemotaxis. These findings were mirrored by increased FN staining in the peritumoural and tumoural areas in vivo. IFN alpha thus determines increased FN production and hence massive local recruitment and activation of fibroblasts, with a modification of the EM. The several activities of IFN alpha should thus be considered prior to its employment in clinical trials.

Adenocarcinoma↗

PubMed1997

Post-translational up-regulation of the cell surface-associated alpha component of the human type I interferon receptor during differentiation of peripheral blood monocytes: role in the biological response to type I interferon.

Human peripheral blood monocytes cultured in vitro exhibit a greater sensitivity to the antiviral effect of type I interferon (IFN) compared to freshly isolated monocytes. We evaluated the effect of macrophage differentiation on the expression of type I IFN receptors (IFN-R). Binding studies with iodinated IFN-alpha 2 and Scatchard plot analysis revealed that a single class of high-affinity receptors was present in freshly isolated monocytes. Monocyte differentiation to macrophages resulted in a three- to fourfold increase in the number of cell surface receptors with no change in their affinity. Polymerase chain reaction analysis of RNA revealed that comparable levels of mRNA for the IFN-R alpha (IFNAR1) and IFNAR2 components were expressed in freshly isolated monocytes and 7-day cultured macrophages. Likewise, the levels of IFNAR1 protein remained constant over time in culture. Immunofluorescence studies revealed that IFNAR1 was localized in intracellular compartments of freshly isolated monocytes, whereas it was predominantly detected on the cell surface in 7-day cultured macrophages. The increased expression of IFN-R on the plasma membrane of cultured macrophages may, at least in part, account for the increased antiviral effect of type I IFN in these cells. These modifications represent one of the events occurring during monocyte differentiation that may play a role in the regulation of macrophage functions.

Adolescent↗

PubMed1997

Induction of cytokines by HIV-1 and its gp120 protein in human peripheral blood monocyte/macrophages and modulation of cytokine response during differentiation.

We previously reported that in vitro culture of human peripheral blood monocytes resulted in a time-dependent differentiation into macrophages and in an enhanced capacity for producing certain cytokines [i.e., tumor necrosis factor alpha, interleukin-6 (IL-6), and interferon-beta (IFN-beta)] in response to bacterial lipopolysaccharide (LPS). HIV-1 infection or gp120 treatment of monocyte/macrophages resulted in the induction of low levels of IFN-beta, which were very effective in restricting viral replication in 7-day cultured macrophages but not in freshly isolated cells. This enhanced response of macrophages was due to a higher sensitivity of these cells to the antiviral effect of IFN-beta. Consistent with this finding, 7-day cultured macrophages exhibited higher levels of type I IFN receptors than 1-day cultured monocytes. Treatment of monocyte/macrophages with gp120 also caused a marked increase in IL-10 secretion, regardless of the differentiation state. No IL-12 secretion was detected in monocyte/macrophage cultures treated with gp120 alone. However, consistent IL-12 secretion was found in 7-day cultured macrophages primed with IFN-beta and subsequently stimulated with gp120. Macrophages responded more efficiently than monocytes to the priming effect of IFN-beta for IL-12 production. This was consistent with a stronger antiviral response against vesicular stomatitis virus by these cells as well as with a higher expression of IFN-beta receptors. The finding that the acquisition of the macrophage phenotype is associated with an increased capacity to respond to environmental signals (such as type I and type II IFNs) underlines the importance of the differentiation process for the selection of a certain repertoire of responses that may allow these cells to have important functions in vivo.

Cell Differentiation↗

PubMed1997

U937-SCID mouse xenografts: a new model for acute in vivo HIV-1 infection suitable to test antiviral strategies.

In this study we attempted to develop a new xenochimeric model for HIV infection in SCID mice, characterized by an easy engraftment of target cells, high levels of viremia and long-lasting HIV-1 infection. SCID mice were injected subcutaneously with uninfected human U937 cells and cell-free HIV-1 (IIIB strain) or HIV-1-infected human peripheral blood lymphocytes (PBL). Mice were evaluated for tumor growth, viral infection at the tumor level (DNA-polymerase chain reaction (PCR), RNA-PCR) and immunostaining for the p55/p18 HIV protein) and p24 antigenemia or serum HIV-1 RNA copies. Pretreatment of mice with antibodies to either mouse-IFN alpha/beta or granulocytes resulted in a tumor take and levels of p24 antigenemia higher than in control mice. In mice treated with these antibody preparations, there was a long-lasting HIV infection with the presence of high levels of circulating infectious virus (serum p24 values up to 4000 pg/ml and serum RNA copies up to 5 x 10(7)/ml over 3 months, with the majority of the cells expressing HIV-antigens at the tumor site). Intraperitoneal treatment of SCID mice with AZT (480 mg/kg per day) resulted in a complete inhibition of both p24 and RNA HIV-1 copies in the serum, together with a marked reduction in the number of infected cells and the levels of virus expression at the tumor site. We conclude that some specific features of this model (i.e. easy establishment, high reproducibility, well defined kinetics of virus infection, massive and long persistent viremia) underline the special advantages of its use for testing new antiviral therapies.

Animals↗

PubMed1997

The biological relevance of polykaryons in the immune response.

Peripheral blood monocyte-derived multinucleated giant cells are a well-known feature of chronic inflammatory conditions. Similarly, virus-induced syncytia derived from CD4+ cells are considered to be typical of human immunodeficiency virus infection under culture conditions. Here, Stefano Fais and colleagues summarize recent experimental results comparing the mechanisms underlying the formation and fate of these two different polykaryons, discussing their putative role in the immune response.

CD4-Positive T-Lymphocytes↗

PubMed1997

Local and systemic antitumor response after combined therapy of mouse metastatic tumors with tumor cells expressing IFN-alpha and HSVtk: perspectives for the generation of cancer vaccines.

In this study, we have evaluated the local versus systemic antitumor response in tumor-bearing mice subjected to a combined therapeutic regimen based on the injection of genetically modified Friend erythroleukemia cells (FLC) producing IFN-alpha and expressing the HSVtk (tk) gene, and we have investigated the host immune mechanisms involved in tumor rejection and development of antitumor immunity. Repeated subcutaneous (s.c.) injections of IFNtk-expressing tumor cells, followed by GCV administration, were effective in counteracting the growth of both contralateral parental tumors as well as visceral metastases, whereas similar treatments with control tk cells (i.e. nonproducing IFN) were ineffective. Morphologic analyses of the homolateral and contralateral tumor tissues and in vivo immunosuppression experiments with specific monoclonal antibodies revealed that both CD4+ and CD8+ T lymphocytes played essential roles in the generation of a definite antitumor response after the combined therapeutic regimen. We have also compared the effectiveness of irradiated versus viable tumor vaccines co-expressing the two genes in the FLC model and in the poorly immunogenic metastasizing TS/A adenocarcinoma tumor system. Repeated injections of high doses of irradiated IFN-alpha-tk-expressing tumor cells followed by GCV administration resulted in the cure of the majority of mice bearing established metastatic tumors, while repeated inoculations of the same number of viable tumor vaccines were much less effective. We conclude that; (1) IFN-alpha is an essential cofactor in the generation of a systemic antitumor immunity following the prodrug-induced tumor cell killing; (2) vaccines co-expressing an autotoxic gene and a cytokine gene may represent promising new tools for the treatment of some cancer patients.

Animals↗

PubMed1997

Human immunodeficiency virus type 1 gp120 stimulates cytomegalovirus replication in monocytes: possible role of endogenous interleukin-8.

Recombinant gp120, but not other human immunodeficiency type 1 (HIV-1) structural proteins, dose-dependently stimulates human cytomegalovirus (HCMV) immediate-early antigen (IEA) expression and infectious virus yield in freshly isolated normal monocytes infected with HCMV. Monoclonal antibodies (MAbs) recognizing the gp120 V3 loop, as well as V3 loop octameric multibranched peptides and antibody to galactocerebroside, but not sCD4, abrogate the gp120 stimulation of IEA expression, suggesting that the effect involves V3 loop-galactocerebroside interaction and is not mediated by CD4. Interleukin 8 (IL-8) gene expression is enhanced in monocytes treated with gp120 at the level of both mRNA and released protein. Exogenous IL-8 could replace gp120 in the stimulation of HCMV infection, while a MAb capable of neutralizing IL-8 activity abrogates the gp120-induced HCMV stimulation. These data indicate that HIV-1 glycoprotein induces stimulation of productive infection of monocytes with HCMV and that such stimulation may be mediated by the upregulation of IL-8 gene expression. This is the first evidence that HIV-1 may affect HCMV replication indirectly, via the interaction of gp120 with the monocyte membrane, in the complete absence of retroviral replication, through the stimulation of IL-8 release. Because in HIV-1-infected individuals, HCMV infection is frequently activated and the levels of circulating IL-8 are enhanced, these findings may be pathogenetically relevant.

Antigens, Viral↗

PubMed1997

Induction of relA(p65) and I kappa B alpha subunit expression during differentiation of human peripheral blood monocytes to macrophages.

We evaluated the expression and DNA binding activity of nuclear factor (NF)-kappa B subunits in human peripheral blood monocytes and in monocyte-derived macrophages (MDMs). Constitutive DNA binding activity consisting of p50 homodimers was detected in nuclear extracts from both cell types. An additional complex composed of p50/RelA(p65) heterodimers appeared only in nuclear extracts from 7-day MDMs. Immunoblot analysis showed that the p50 subunit was constitutively expressed in monocytes and MDMs. In contrast, the RelA(p65) subunit was barely detectable in monocytes, but its level increased markedly in MDMs. Analysis of RelA(p65) mRNA revealed that the stability of RelA(p65) mRNA was significantly higher in MDMs, compared with monocytes. In MDMs, an upregulation of I kappa B alpha synthesis as well as the appearance of a novel M(r) 40,000 form of I kappa B alpha were also observed. These results suggest that macrophage differentiation results in the expression of active p50/RelA(p65) heterodimers with the capacity to activate target gene expression. The parallel induction of I kappa B alpha synthesis may allow for the continuous presence of a cytoplasmic reservoir of p50/RelA(p65) complexes that are readily available for inducer-mediated stimulation.

Adolescent↗

PubMed1996

The neglected role of type I interferon in the T-cell response: implications for its clinical use.

Originally described as an antiviral substance, type I interferon (IFN) was subsequently shown to exert multiple biological effects and is now the most frequently used cytokine in the treatment of some viral and neoplastic diseases. Although early studies described various effects on the immune system, the role of type I IFN as an immunoregulatory molecule has long been neglected. Here, Filippo Belardelli and Ion Gresser summarize recent experimental results on the interactions of type I IFN with T cells, which may prove important in its use in patients.

Animals↗

PubMed1996

Cure of mice with established metastatic friend leukemia cell tumors by a combined therapy with tumor cells expressing both interferon-alpha 1 and herpes simplex thymidine kinase followed by ganciclovir.

Transduction of the murine interferon-alpha (IFN-alpha) gene into various malignant mouse tumor cells has resulted in the loss of tumorigenicity and an acquired capacity to induce long-lasting antitumor immunity following their injection into immunocompetent syngeneic mice. In the present study, we investigated the effectiveness of IFN-alpha-producing tumor cells in the therapy of mice with established mouse tumors. In DBA/2 mice bearing subcutaneous (s.c.) Friend erythroleukemia cell (FLC) tumors, we found that to achieve some antitumor response (i) it was necessary to inject high numbers of IFN-alpha-producing FLC, which occasionally lead to the formation of slowly growing tumors; and, that (ii) repeated injections of irradiated IFN-alpha-FLC did not result in any antitumor effect. The therapeutic potential of IFN-alpha-producing FLC rendered sensitive to ganciclovir (GCV), by transfer of the herpes simplex virus thymidine kinase (tk) gene, was investigated. Complete tumor rejection and cure was observed in > or = 70% of the animals after injection of high numbers (10(7)) of IFN-alpha-producing tk-expressing tumor cells followed 4 days later by repeated GCV treatments, whereas only a slight increase in survival time was obtained after administration of control tk-expressing tumor cells (not producing IFN) and GCV. Tumor rejection was associated with a dramatic destruction of tumor tissue and with the subsequent development of a potent and long-lasting antitumor immunity. No therapeutic effect was observed in immunosuppressed nude mice. These data indicate that this approach may represent an effective and safe therapeutic strategy for antitumor cytokine gene therapy.

Animals↗

PubMed1996

T-cell dysfunctions in hu-PBL-SCID mice infected with human immunodeficiency virus (HIV) shortly after reconstitution: in vivo effects of HIV on highly activated human immune cells.

The state of activation of the immune system may be an important factor which renders a host more receptive to human immunodeficiency virus (HIV) and more vulnerable to its effects. To explore this issue with a practical in vivo model, we developed a modified protocol of HIV infection in hu-PBL-SCID mice. First, we assessed the time course of activation of human peripheral blood lymphocytes (hu-PBL) in the peritoneal cavity of SCID mice. At 2 to 24 h after the intraperitoneal injection into SCID mice, there was a clear-cut increase in the percentage of hu-PBL expressing early activation markers (CD69), concomitant with the release of soluble intercellular adhesion molecule-1 (sICAM-1) and the soluble interleukin-2 receptor (sIL-2R) and with the accumulation of mRNAs for a number of human cytokines. At 2 weeks, virtually all of the hu-PBL expressed the memory phenotype (CD45RO) and HLA-DR antigens as well. Cells collected from the SCID mouse peritoneum at 2 and 24 h after transplantation were fully susceptible to in vitro infection with HIV type 1 (HIV-1) in the absence of either IL-2 or mitogens. The injection of HIV into hu-PBL-SCID mice at 2 h after reconstitution resulted in a generalized and productive HIV infection of the xenochimeras. This early HIV-1 infection resulted in a dramatic depletion of human CD4+ cells and in decreased levels of sICAM-1 (in the peritoneal lavage fluid) as well as of sIL-2R and immunoglobulins M and A (in the serum). Enzyme-linked immunosorbent assay and/or reverse transcriptase PCR analysis showed higher levels of IL-4, IL-5, and IL-10 in the HIV-infected animals than in control hu-PBL-SCID mice, while gamma interferon levels in the two groups were comparable. When we compared the current model of HIV-1 infection at 2 weeks after the intraperitoneal injection of the hu-PBL in the SCID mice with the model described here, we found that the majority of immune dysfunctions induced in the 2-h infection of the xenochimeras are not inducible in the 2-week infection. This supports the concept that the state of activation of human cells at the moment of the in vivo infection with HIV-1 is a crucial factor in determining the immune derangement observed in AIDS patients. These results show that some immunological dysfunctions induced by HIV infection in AIDS patients can be mimicked in this xenochimeric model. Thus, the hu-PBL-SCID mouse model may be useful in exploring, in vivo, the relevance of hu-PBL activation and differentiation in HIV-1 infection and for testing therapeutic intervention directed towards either the virus or the immune system.

Animals↗

PubMed1996

Induction of interleukin-12 (IL-12) by recombinant glycoprotein gp120 of human immunodeficiency virus type 1 in human monocytes/macrophages: requirement of gamma interferon for IL-12 secretion.

We studied the effects of the gp120 glycoprotein of human immunodeficiency virus type 1 on the expression of interleukin-12 (IL-12) in human monocytes and in monocyte-derived macrophages. Induction of the mRNA for both the p35 and p40 subunits of IL-12 was observed in both cell types after gp120 treatment. We then evaluated cytokine secretion by using an enzyme-linked immunosorbent assay which recognizes only the IL-12 heterodimer. No IL-12 was detected in monocytes/macrophages treated with gp120 alone. A consistent IL-12 secretion was found in macrophages primed with gamma interferon (IFN-gamma) and subsequently treated with gp120. Low levels of IL-12 were occasionally observed in IFN-gamma-primed monocytes stimulated with gp120. The greater response of macrophages than of monocytes to the priming effect of IFN-gamma was consistent with the finding that IFN-gamma induced a much stronger antiviral state to vesicular stomatitis virus in macrophages than in monocytes. These data indicate that gp120 is an inducer of IL-12 expression in monocytes/macrophages and that IFN-gamma is an essential cofactor for IL-12 secretion, especially in differentiated macrophages.

Base Sequence↗