Biomedical subjects

Angela Santoni

Publications and source records attributed to Angela Santoni.

At least 19 recordsLinked to original sources

PubMed2007

Human immunodeficiency virus 1 Nef protein downmodulates the ligands of the activating receptor NKG2D and inhibits natural killer cell-mediated cytotoxicity.

Natural killer (NK) cells are a major component of the host innate immune defence against various pathogens. Several viruses, including Human immunodeficiency virus 1 (HIV-1), have developed strategies to evade the NK-cell response. This study was designed to evaluate whether HIV-1 could interfere with the expression of NK cell-activating ligands, specifically the human leukocyte antigen (HLA)-I-like MICA and ULBP molecules that bind NKG2D, an activating receptor expressed by all NK cells. Results show that the HIV-1 Nef protein downmodulates cell-surface expression of MICA, ULBP1 and ULBP2, with a stronger effect on the latter molecule. The activity on MICA and ULBP2 is well conserved in Nef protein variants derived from HIV-1-infected patients. In HIV-1-infected cells, cell-surface expression of NKG2D ligands increased to a higher extent with a Nef-deficient virus compared with wild-type virus. Mutational analysis of Nef showed that NKG2D ligand downmodulation has structural requirements that differ from those of other reported Nef activities, including HLA-I downmodulation. Finally, data demonstrate that Nef expression has functional consequences on NK-cell recognition, causing a decreased susceptibility to NK cell-mediated lysis. These findings provide a novel insight into the mechanisms evolved by HIV-1 to escape from the NK-cell response.

Cell Line↗

PubMed2006

Oxidative stress inhibits IFN-alpha-induced antiviral gene expression by blocking the JAK-STAT pathway.

BACKGROUND/AIMS: Unresponsiveness to IFN-alpha is common in chronic hepatitis C. Since conditions associated with an increased oxidative stress (advanced age, steatosis, fibrosis, iron overload, and alcohol consumption) reduce the likelihood of response, we hypothesized that oxidative stress may affect the antiviral actions of IFN-alpha. METHODS: We examined in a human hepatocellular carcinoma cell line (Huh-7) the effect of hydrogen peroxide (H2O2), as a generator of oxidative stress, on the IFN-alpha signaling pathway. RESULTS: Pretreatment of Huh-7 cells with 0.5-1 mM H2O2 resulted in the suppression of the IFN-alpha-induced antiviral protein MxA and of IRF-9 mRNA expression. The reduced expression of these genes was associated to H2O2 -mediated suppression of the IFN-alpha-induced assembly of signal transducer and activator of transcription (STAT) factors to specific promoter motifs on IFN-alpha-inducible genes. This was accomplished by preventing the IFN-alpha-induced tyrosine phosphorylation of STAT-1 and STAT-2 through the inactivation of the upstream receptor associated tyrosine kinases, JAK-1 and Tyk-2. The suppression was fast, occurring within 5mins of pretreatment with H2O2, and did not require protein synthesis. CONCLUSIONS: In conclusion, oxidative stress impairs IFN-alpha signaling and might cause resistance to the antiviral action of IFN-alpha in chronically HCV infected patients with high level of oxidative stress in the liver.

Blotting, Western↗

PubMed2006

The cytoskeleton regulatory protein hMena (ENAH) is overexpressed in human benign breast lesions with high risk of transformation and human epidermal growth factor receptor-2-positive/hormonal receptor-negative tumors.

PURPOSE: hMena (ENAH), a cytoskeleton regulatory protein involved in the regulation of cell motility and adhesion, is overexpressed in breast cancer. The aim of this study was to define at what stage of breast carcinogenesis hMena is overexpressed and to correlate hMena overexpression with established prognostic factors in breast cancer, focusing on human epidermal growth factor receptor-2 (HER-2). EXPERIMENTAL DESIGN: hMena expression was assessed immunohistochemically in a prospective cohort of cases (n = 360) encompassing a highly representative spectrum of benign breast diseases associated with different risk of transformation, in situ, invasive, and metastatic tumors. Correlations with conventional pathologic and prognostic variables, such as proliferation index, hormonal receptor status, and HER-2 overexpression, were also evaluated. In vitro experiments were done to study the effect of neuregulin-1 and Herceptin treatments on hMena expression. RESULTS: hMena protein is undetectable in normal breast and is weakly expressed in a small percentage of low-risk benign diseases (9%), but displays a progressive and significant increase of positivity in benign lesions at higher risk of transformation (slightly increased risk 43%; moderate increased risk 67%), in in situ (72%), invasive (93%), and metastatic breast cancer (91%). A significant direct correlation with tumor size (P = 0.04), proliferation index (P < 0.0001), and HER-2 overexpression (P < 0.0001) and an inverse relationship with estrogen (P = 0.036) and progesterone receptors (P = 0.001) are found in invasive carcinomas. In vitro experiments show that neuregulin-1 up-regulates, whereas Herceptin down-regulates, hMena expression. CONCLUSIONS: Our data provide new insights into the relevance of actin-binding proteins in human breast carcinogenesis and indicate hMena overexpression as a surrogate indicator in breast disease management.

Adolescent↗

PubMed2006

High-efficient lentiviral vector-mediated gene transfer into primary human NK cells.

OBJECTIVE: The long-term transfection of genes into primary natural killer (NK) cells without disrupting normal cellular functions has been proven to be difficult with currently available gene-transfer methods. In this study, we establish a lentiviral vector-based technique for improved gene transfer into human NK cells in vitro and we report on high-efficient transduction of freshly isolated as well as cultured primary NK cells. METHODS: Freshly isolated or primary cultured human NK cells, as well as the human NK cell line YTS, were transduced with replication-incompetent human immunodeficiency virus (HIV)-based lentiviral vector bearing a GFP reporter gene or a gene of interest under the control of the elongation factor 1alpha (EF1alpha) promoter. Transduction efficiencies were monitored by flow cytometry. RESULTS: A long-term transgene expression was detected in up to 98% of YTS NK cells, whereas in freshly isolated or primary cultured NK cells exposed to interleukin (IL)-2 plus IL-12 upon infection, efficiency was in the range of 50% to 90%. Moreover, in freshly isolated quiescent NK cells a transfection efficiency of 18% to 20% was achieved without stimulation. Notably, no major phenotypic and functional modifications were observed in transduced cells with respect to control cells: the expression levels of activating receptors, CD69-antigen induction as well as cytotoxic function were unaffected. CONCLUSION: Results of our study demonstrate that NK cells can be efficiently transduced by lentiviral vectors.

Antigens, CD↗

PubMed2006

Recognition of a carbohydrate xenoepitope by human NKRP1A (CD161).

BACKGROUND: Many immunologically important interactions are mediated by leukocyte recognition of carbohydrates via cell surface receptors. Uncharacterized receptors on human natural killer (NK) cells interact with ligands containing the terminal Galalpha(1,3)Gal xenoepitope. The aim of this work was to isolate and characterize carbohydrate binding proteins from NK cells that bind alphaGal or other potential xenoepitopes, such as N-acetyllactosamine (NAcLac), created by the deletion of alpha1,3galactosyltransferase (GT) in animals. METHODS AND RESULTS: Initial analysis suggested the human C-type lectin NKRP1A bound to a pool of glycoconjugates, the majority of which contained the terminal Galalpha(1,3)Gal epitope. This was confirmed by high level binding of cells expressing NKRP1A to mouse laminin, which contains a large number of N-linked oligosaccharides with the Galalpha(1,3)Gal structure. The consequence of removing the terminal alphaGal was then investigated. Elevated NAcLac levels were observed on thymocytes from GT-/- mice. Exposing NAcLac on laminin, by alpha-galactosidase treatment, resulted in a significant increase in NKRP1A binding. CONCLUSIONS: NKRPIA binds to the alphaGal epitope. Moreover, exposing NAcLac by removal of alphaGal resulted in an increase in binding. This may be relevant in the later phases of xenotransplant rejection if GT-/- pigs, like GT-/- mice, display increased NAcLac expression.

Amino Sugars↗

PubMed2005

CIN85 regulates the ligand-dependent endocytosis of the IgE receptor: a new molecular mechanism to dampen mast cell function.

Ligation of the high-affinity receptor for IgE (Fc epsilonRI), constitutively expressed on mast cells and basophils, promotes cell activation and immediate release of allergic mediators. Furthermore, Fc epsilonRI up-regulation on APC from atopic donors is involved in the pathophysiology of allergic diseases. In consideration of the clinical relevance of the IgE receptor, the down-modulation of Fc epsilonRI expression in mast cells may represent a potential target for handling atopic diseases. In an effort to identify new molecular mechanisms involved in attenuating Fc epsilonRI expression and signaling, we focused our attention on CIN85, a scaffold molecule that regulates, in concert with the ubiquitin ligase Cbl, the clathrin-mediated endocytosis of several receptor tyrosine kinases. In the present study, we show that endogenous CIN85 is recruited in Cbl-containing complexes after engagement of the Fc epsilonRI on a mast cell line and drives ligand-induced receptor internalization. By confocal microscopic analysis, we provide evidence that CIN85 directs a more rapid receptor sorting in early endosomes and delivery to a lysosomal compartment. Furthermore, biochemical studies indicate that CIN85 plays a role in reducing the expression of receptor complex. Finally, we demonstrate that CIN85-overexpressing mast cells are dramatically impaired in their ability to degranulate following Ag stimulation, suggesting that the accelerated internalization of activated receptors by perturbing the propagation of Fc epsilonRI signaling may contribute to dampen the functional response. This role of CIN85 could be extended to include other multimeric immune receptors, such as the T and B cell receptors, providing a more general molecular mechanism for attenuating immune responses.

Animals↗

PubMed2005

CD8 cell division maintaining cytotoxic memory occurs predominantly in the bone marrow.

Long-term persistence of Ag-experienced CD8 cells, a class of T lymphocytes with cytotoxic function, contributes to immunological memory against intracellular pathogens. After Ag clearance, memory CD8 cells are maintained over time by a slow proliferation, primarily cytokine driven. In this article, we show that the bone marrow (BM) is the crucial organ where such basal division of memory CD8 cells occurs. BM memory CD8 cells contain a higher percentage of proliferating cells than their corresponding cells in either spleen or lymph nodes from C57BL/6 mice. This occurs both in the case of memory-phenotype CD44(high) CD8 cells and in the case of Ag-specific memory CD8 cells. Importantly, the absolute number of Ag-specific memory CD8 cells dividing in the BM largely exceeds that in spleen, lymph nodes, liver, and lung taken together. In the BM, Ag-specific memory CD8 cells express lower levels of CD127, i.e., the alpha-chain of IL-7R, than in either spleen or lymph nodes. We interpret these results as indirect evidence that Ag-specific memory CD8 cells receive proliferative signals by IL-7 and/or IL-15 in the BM and propose that the BM acts as a saturable "niche" for the Ag-independent proliferation of memory CD8 cells. Taken together, our novel findings indicate that the BM plays a relevant role in the maintenance of cytotoxic T cell memory, in addition to its previously described involvement in long-term Ab responses.

Adoptive Transfer↗

PubMed2005

Chemoattractant induces LFA-1 associated PI 3K activity and cell migration that are dependent on Fyn signaling.

The activation state of integrins on leukocytes is tightly controlled by the association of intracellular molecules to the cytoplasmic domain of the integrin. To identify signaling intermediates involved in chemoattractant receptor-induced integrin activation, we analyzed the ability of LFA-1 integrin to associate with members of Src tyrosine kinase and phosphatidylinositol 3Kinase (PI 3K) families following fMLP stimulation of a lymphoid T cell line stably expressing the fMLP receptor (fPR). fMLP-induced cell activation resulted in a rapid increase of integrin-associated PI 3Kinase activity in G protein and Src kinase dependent manner. We show, upon fMLP-stimulation, a rapid and transient association of the Src tyrosine kinase Fyn with LFA-1. Also, by transiently expressing mutant forms of this kinase, we demonstrated that Fyn is required for the integrin associated PI 3K activity. Furthermore, overexpression of the mutant form of Fyn resulted in a significant impairment of fMLP-induced cell migration through ICAM-1-coated filters, while ICAM-1 independent migration was not affected. Our observations indicate that chemoattractant receptor engagement induces Fyn-dependent PI 3K activation in association with LFA-1 and suggests that Fyn is necessary to initiate and/or to regulate chemoattractant-mediated LFA-1 activation to promote directional migration.

Animals↗

PubMed2005

Arf6: a new player in FcgammaRIIIA lymphocyte-mediated cytotoxicity.

The activation of phosphoinositide metabolism represents a critical step in the signaling pathways leading to the activation of cytolytic machinery, but its regulation is partially understood. We report here that the stimulation of the low-affinity receptor for immunoglobulin G (IgG) (FcgammaRIIIA, CD16) on primary human natural killer (NK) cells induces a phosphatidylinositol 3-kinase (PI3K)-dependent activation of the small G protein Arf6. We first demonstrate a functional role for Arf6-dependent signals in the activation of the antibody-dependent cellular cytotoxicity (ADCC) attributable to the control of secretion of lytic granule content. We also show that Arf6 couples CD16 to the lipid-modifying enzymes phosphatidylinositol4phosphate 5-kinase type I alpha (PI5KIalpha) and phospholipase D (PLD) that are involved in the control of granule secretion; Arf6, but not Rho family small G proteins RhoA and Rac1, is required for receptor-induced PI5KIalpha membrane targeting as well as for PI5KIalpha and PLD activation. Our findings suggest that Arf6 plays a crucial role in the generation of a phosphatidylinositol4,5-bisphosphate (PIP2) plasma membrane pool required for cytolytic granule-mediated target cell killing.

ADP-Ribosylation Factor 6↗

PubMed2005

Hyperthermia enhances CD95-ligand gene expression in T lymphocytes.

Hyperthermia represents an interesting therapeutic strategy for the treatment of tumors. Moreover, it is able to regulate several aspects of the immune response. Fas (APO-1/CD95) and its ligand (FasL) are cell surface proteins whose interaction activates apoptosis of Fas-expressing targets. In T cells, the Fas-Fas-L system regulates activation-induced cell death, is implicated in diseases in which lymphocyte homeostasis is compromised, and plays an important role during cytotoxic and regulatory actions mediated by these cells. In this study we describe the effect of hyperthermia on activation of the fas-L gene in T lymphocytes. We show that hyperthermic treatment enhances Fas-L-mediated cytotoxicity, fas-L mRNA expression, and fas-L promoter activity in activated T cell lines. Our data indicate that hyperthermia enhances the transcriptional activity of AP-1 and NF-kappaB in activated T cells, and this correlates with an increased expression/nuclear translocation of these transcription factors. Moreover, we found that heat shock factor-1 is a transactivator of fas-L promoter in activated T cells, and the overexpression of a dominant negative form of heat shock factor-1 may attenuate the effect of hyperthermia on fas-L promoter activity. Furthermore, overexpression of dominant negative mutants of protein kinase Cepsilon (PKCepsilon) and PKCtheta; partially inhibited the promoter activation and, more importantly, could significantly reduce the enhancement mediated by hyperthermia, indicating that modulation of PKC activity may play an important role in this regulation. These results add novel information on the immunomodulatory action of heat, in particular in the context of its possible use as an adjuvant therapeutic strategy to consider for the treatment of cancer.

Animals↗

PubMed2005

NK cell regulation of T cell-mediated responses.

NK cells promote adaptive immune responses through their production of type 1 and type 2 cytokines or chemokines. Secretion of these factors by activated NK cells influences the differentiation of B and T lymphocytes. Increasing evidence indicates that NK cells are also directly involved in dendritic cell (DC) maturation. By contrast, a potential role for direct cell-cell interactions between NK and T lymphocytes, in particular CD4(+) T cells, has not been explored. We provide evidence that activated human NK cells are able of promoting TcR-dependent proliferation of resting autologous peripheral blood CD4(+) T cells by a process that involves costimulatory molecules of the immunoglobulin (Ig) and tumor necrosis factor (TNF) superfamilies. These findings suggest a novel link between natural and adaptative immune responses.

Cell Communication↗

PubMed2004

Cross-talk between activated human NK cells and CD4+ T cells via OX40-OX40 ligand interactions.

It is important to understand which molecules are relevant for linking innate and adaptive immune cells. In this study, we show that OX40 ligand is selectively induced on IL-2, IL-12, or IL-15-activated human NK cells following stimulation through NKG2D, the low affinity receptor for IgG (CD16) or killer cell Ig-like receptor 2DS2. CD16-activated NK cells costimulate TCR-induced proliferation, and IFN-gamma produced by autologous CD4+ T cells and this process is dependent upon expression of OX40 ligand and B7 by the activated NK cells. These findings suggest a novel and unexpected link between the natural and specific immune responses, providing direct evidence for cross-talk between human CD4+ T cells and NK receptor-activated NK cells.

Antigens, CD↗

PubMed2004

p38 MAPK activation controls the TLR3-mediated up-regulation of cytotoxicity and cytokine production in human NK cells.

Natural killer (NK) cells are a component of the innate immunity against viral infections through their rapid cytotoxic activity and cytokine production. Although the synthetic double-stranded (ds) RNA polyinosinic-polycytidylic acid (poly I:C), a mimic of a common product of viral infections, is known to rapidly up-regulate their in vivo functions, NK cell ability to directly respond to dsRNA is still mostly unknown. Our results show that treatment with poly I:C significantly up-regulates both natural and CD16-mediated cytotoxicity of highly purified human NK cells. Poly I:C also induces the novel capability of producing CXCL10 chemokine in human NK cells and synergistically enhances interferon-gamma (IFN-gamma) production induced by either adaptive or innate cytokines. In accordance with the expression of Toll-like receptor-3 (TLR3) and of TRIF/TICAM-1 adaptor, poly I:C stimulation induces the activation of interferon regulatory factor-3 (IRF-3) transcription factor and of p38 mitogen-activated protein kinase (MAPK) in human NK cells. Finally, we demonstrate that p38 MAPK activity is required for the dsRNA-dependent enhancement of cytotoxicity and CXCL10 production. The occurrence of dsRNA-induced signaling and functional events closely correlates with the TLR3 mRNAprofile in different NK cell populations. Taken together, these data identify p38 as a central component of NK cell ability to directly respond to dsRNA pathogen-associated molecular pattern (PAMP).

Adaptor Proteins, Vesicular Transport↗

PubMed2004

Noncompetitive allosteric inhibitors of the inflammatory chemokine receptors CXCR1 and CXCR2: prevention of reperfusion injury.

The chemokine CXC ligand 8 (CXCL8)/IL-8 and related agonists recruit and activate polymorphonuclear cells by binding the CXC chemokine receptor 1 (CXCR1) and CXCR2. Here we characterize the unique mode of action of a small-molecule inhibitor (Repertaxin) of CXCR1 and CXCR2. Structural and biochemical data are consistent with a noncompetitive allosteric mode of interaction between CXCR1 and Repertaxin, which, by locking CXCR1 in an inactive conformation, prevents signaling. Repertaxin is an effective inhibitor of polymorphonuclear cell recruitment in vivo and protects organs against reperfusion injury. Targeting the Repertaxin interaction site of CXCR1 represents a general strategy to modulate the activity of chemoattractant receptors.

Allosteric Regulation↗

PubMed2004

Ras inhibition amplifies cisplatin sensitivity of human glioblastoma.

Resistance to chemotherapy is a common feature of malignant gliomas. This resistance is mediated by receptor tyrosine kinase (RTK)-regulated signaling. p21-Ras protein is pivotal in the propagation of the signal originated from many RTKs. Our aim was to investigate whether inhibition of Ras pathway affects the response to cisplatin in malignant gliomas. We found an enhanced sensitivity to cisplatin of two glioblastoma cell lines expressing dominant negative Ras. Moreover, DN-Ras expressing cells, implanted in nude mice, resulted in being extremely sensitive to cisplatin. The growth of all the tumors was significantly inhibited by combining DN-Ras adenovirus infection with cisplatin treatment. The majority of glioma cells expressing DN-Ras underwent apoptosis in response to cisplatin. In vivo, DN-Ras alone did not influence the growth of tumors, suggesting that the effects of Ras-inhibition observed in vitro could not be extrapolated in vivo. The survival signal pathway transduced by Ras was essentially mediated by inhibition of caspase-9 cleavage via PI3K/Akt.

Adenoviridae↗

PubMed2004

Human Mena protein, a serex-defined antigen overexpressed in breast cancer eliciting both humoral and CD8+ T-cell immune response.

Screening of a cDNA expression library from a primary breast tumor with the autologous patient serum led to the isolation of 6 cDNA clones corresponding to 3 different genes, including a novel gene that maps to chromosome 1 and encodes the human homologue of mouse Mena (hMena, cDNA clone RMNY-BR-55), a protein of the Ena/VASP family involved in the regulation of cell motility and adhesion. A cancer-restricted antibody response against hMena was demonstrated, since 18/93 cancer patient sera, the majority (10/52) from breast cancer, showed anti-hMena-specific IgG, while no antibodies were present in healthy donors. When hMena protein expression was analyzed by Western blot and immunohistochemistry, the antigen was overexpressed in the majority of breast cancer cell lines and in 75% of primary breast tumor lesions evaluated. Furthermore, when HLA-A2-restricted peptides from the hMena sequence were used to stimulate CD8+ T cells, an hMena-specific response was found in 9 out of 12 HLA-A2+ breast cancer patients. In 4 patients, this cell-mediated immune response was concomitant with antibody response to hMena. Furthermore, an hMena-specific T-cell line was established from an HLA-A2+ breast cancer patient whose primary tumor lesion overexpressed the hMena protein. The present findings highlight the emerging role that overexpression of cytoskeleton regulatory components may have in the induction of a specific antitumor immune response.

Antibodies, Neoplasm↗

PubMed2004

Impaired natural and CD16-mediated NK cell cytotoxicity in patients with WAS and XLT: ability of IL-2 to correct NK cell functional defect.

In this study we show that Wiskott-Aldrich syndrome protein (WASp), a critical regulator of actin cytoskeleton that belongs to the Scar/WAVE family, plays a crucial role in the control of natural killer (NK) cell cytotoxicity. Analysis of NK cell numbers and cytotoxic activity in patients carrying different mutations in the WASP coding gene indicated that although the percentage of NK cells was normal or increased, natural cytotoxicity and antibody-mediated NK cell cytotoxicity were inhibited in all patients with the classical WAS phenotype and in most patients carrying mutations associated with the X-linked thrombocytopenia (XLT) phenotype. The inhibition of NK cell-mediated cytotoxicity was associated with the reduced ability of WAS and XLT NK cells to form conjugates with susceptible target cells and to accumulate F-actin on binding. Treatment with interleukin-2 (IL-2) corrected the functional defects of NK cells by affecting their ability to bind to sensitive target cells and to accumulate F-actin. In addition, we provide information on the molecular mechanisms that control WASp function, demonstrating that binding of NK cells to sensitive targets or triggering through CD16 by means of reverse antibody-dependent cellular cytotoxicity (ADCC) rapidly activates Cdc42. We also found that WASp undergoes tyrosine phosphorylation upon CD16 or beta2-integrin engagement on NK cells.

Actins↗

PubMed2004

Key role of proline-rich tyrosine kinase 2 in interleukin-8 (CXCL8/IL-8)-mediated human neutrophil chemotaxis.

The signalling pathways leading to CXCL8/IL-8-induced human neutrophil migration have not been fully characterized. The present study demonstrates that CXCL8 induces tyrosine phosphorylation as well as enzymatic activity of proline-rich tyrosine kinase 2 (Pyk2), a non-receptor protein tyrosine kinase (PTK), in human neutrophils. Induction of Pyk2 tyrosine phosphorylation by CXCL8 is regulated by Src PTK activation, whereas it is unaffected by phosphatidylinositol 3-kinase activation. Inhibition of Pyk2 activation by PP1, a Src PTK inhibitor, is paralleled by the inhibition of CXCL8-mediated neutrophil chemotaxis. Among CXCL8 receptors, Src protein tyrosine kinase activation selectively regulates CXCR1-mediated polymorphonuclear neutrophil (PMN) chemotaxis. Overexpression of PykM, the kinase-dead mutant of Pyk2, blocks CXCL8-induced chemotaxis of HL-60-derived PMN-like cells, thus pinpointing the key role of Pyk2 in CXCL8-induced chemotaxis.

Cells, Cultured↗