Biomedical subjects

P Rovere

Publications and source records attributed to P Rovere.

At least 37 records · Page 2Linked to original sources

PubMed2000

Caspase inhibition reveals functional cooperation between p55- and p75-TNF receptors in cell necrosis.

TNF-induced caspase activation is critically involved in both apoptosis and protection from cell necrosis. We have investigated the roles of the p55- and p75-TNF receptors (TNFR1 and TNFR2) in the induction of mouse L-M cell death in the presence of a caspase inhibitor (zVAD-fmk) and a transcription inhibitor (actinomycin D), i.e. under conditions in which protective pathways requiring caspase activation and protein synthesis were blocked. Cytometric analysis after TNF treatment showed that apoptosis was inhibited, while necrosis was highly activated. In contrast, apoptosis was observed in cells treated with TNF and actinomycin D alone. Stimulation of TNFR1 was sufficient to induce either cell necrosis or apoptosis, even when we blocked endogenous TNF with an anti-murine TNF antibody. Experiments based on the use of receptor-agonist and antagonist antibodies also showed that TNFR2 contributes to cell necrosis and apoptosis. Simultaneous stimulation of TNFR2 and TNFR1 with specific agonists indicated that TNFR2 functionally cooperates with TNFR1 to potentiate the response indirectly, by inducing endogenous TNF cytotoxicity. Caspase inhibitors enhanced the cytotoxic effect of endogenous TNF, suggesting that TNFR2 modulation can regulate the global necrotic response to TNF. TNFR2 modulation could play an important role in determining the response to TNF in pathophysiological conditions characterized by caspase down-regulation and local TNF production.

Amino Acid Chloromethyl Ketones↗

PubMed1999

Generation of nitric oxide by the inducible nitric oxide synthase protects gamma delta T cells from Mycobacterium tuberculosis-induced apoptosis.

Gamma delta T cells are early recruited into mycobacterial lesions. Upon microbial Ag recognition, gamma delta cells secrete cytokines and chemokines and undergo apoptosis via CD95/CD95 ligand (CD95L) interaction, possibly influencing the outcome of infection and the characteristics of the disease. In this paper we show that activated phagocytes acquire, upon challenge with Mycobacterium tuberculosis, the ability to inhibit M. tuberculosis-induced gamma delta cell apoptosis. Apoptosis protection was due to NO because it correlated with NO synthase (NOS)-2 induction and activity in scavenger cells and was abrogated by NOS inhibitors. Furthermore, the NO donor S-nitrosoacetylpenicillamine mimicked the effect of enzyme induction. NO left unaffected the expression of CD95 and CD95L, suggesting interference with an event ensuing CD95/CD95L interaction. NO was found to interfere with the intracellular accumulation of ceramide and the activation of caspases, which were involved in gamma delta T cells apoptosis after M. tuberculosis recognition. We propose that NO generated by infected macrophages determines the life span and therefore the function of lymphocytes at the infection site, thus linking innate and adaptive immunity.

Animals↗

PubMed1999

Immunogenicity of apoptotic cells in vivo: role of antigen load, antigen-presenting cells, and cytokines.

Apoptosis allows the clearance of unwanted cells from living tissues without causing inflammation. Processing of phagocytosed apoptotic cells yields Ags that access the cytosol and the MHC class I pathway of engulfing cells and are recognized by Ag-specific CTL. We show here that injection of apoptotic RMA cells, a syngeneic T cell lymphoma, into C57BL/6 mice results in priming of a functional and long-lasting tumor-specific immune response. Cross-priming of CTLs by apoptotic cells requires CD4+ T cell help. Apoptotic cells, however, are at least 20-fold less immunogenic than nonreplicating live cells. Immunogenicity of apoptotic cells is proportional to the number of cells injected, correlates with the serum concentration of IL-10 and IL-1beta cytokines, and is enhanced in IL-10 knockout mice. Moreover, immunization with dendritic cells (DCs), but not macrophages (Mphi), pulsed with apoptotic cells primes tumor-specific CTLs and confers protection against a tumor challenge. Our findings demonstrate that tumor cells undergoing apoptosis are, though scarcely, immunogenic in vivo, outline the different roles of Mphi and DCs in the physiologic clearance of unwanted cells, and have implications in designing immunomodulating vaccines.

Adoptive Transfer↗

PubMed1999

Blockade of the Fas-triggered intracellular signaling pathway in human melanomas is circumvented by cytotoxic lymphocytes.

Fas and Fas ligand (FasL) have been found both in lymphoid and in non-lymphoid malignancies, and are thought to play a role in the interplay between tumors and the immune system. Here we investigated Fas/FasL expression, function and intracellular signalling pathways in human melanomas. Of 5 melanoma cell lines, 3 expressed Fas at their surface, and all of them expressed FasL. FasL was functional, since it triggered Fas-induced apoptosis of human T lymphocytes clones. Conversely, cross-linking of Fas molecule with a specific monoclonal antibody failed to induce apoptosis in any of the melanomas tested, or ceramide intracellular accumulation or caspase-3 activation, pointing to an early alteration in the Fas-triggered signaling cascade. All melanomas retained the ability to undergo apoptosis induced by cytotoxic lymphocytes, which was mediated by the granule exocytosis mechanism. This suggests that melanoma cells evade immune-mediated Fas-triggered apoptosis via a selective blockade of the Fas apoptotic pathway. Cytotoxic lymphocytes, however, may circumvent tumor resistance to Fas-induced death via granzyme-mediated apoptosis, further supporting the development of immunotherapeutic strategies in the treatment of cancer.

Antigens, CD↗

PubMed1999

Medullary thyroid carcinomas in transgenic mice expressing a Polyoma carboxyl-terminal truncated middle-T and wild type small-T antigens.

Medullary thyroid carcinoma (MTC) is a rare human tumor affecting the calcitonin-secreting c-cells of the thyroid. Here we report that two independent strains of transgenic mice expressing a Polyomavirus (Py) truncated middle-T antigen (deltaMT), consisting of the amino-terminal 304 amino acids, and the full length Py small-T antigen, developed multifocal bilateral MTCs with 100% penetrance. Occasionally one strain also developed mammary and bone tumors. Furthermore, offspring from both transgenic lines displayed pronounced waviness of the whiskers and fur, previously associated with defective epidermal growth factor receptor signaling. Transgene transcription, driven by the homologous early promoter/enhancer, and the corresponding translation products were detected in tumors and in many other organs which did not develop pathologies. The subcellular distribution of deltaMT and its interactions with the adapter proteins of the SHC family have also been analysed. Our study describes a novel murine model of MTC and provides evidence that the N-terminal 304 amino acid fragment of Py middle-T antigen, possibly in co-operation with small-T antigen, acts as a potent oncogene in c-cells of the thyroid.

Animals↗

PubMed1999

Engagement of B cell receptor regulates the invariant chain-dependent MHC class II presentation pathway.

The intracellular sites in which Ags delivered by the B cell receptor (BCR) are degraded and loaded onto class II molecules remain poorly defined. To address this issue, we generated wild-type and invariant chain (Ii)-deficient H-2k mice bearing BCR specific for hen egg lysozyme. Our results show that, 1) unlike Ags taken up from the fluid phase, Ii is required for presentation of hen egg lysozyme internalized through the BCR in a manner independent of the peptide analyzed; 2) BCR ligation induces intracellular accumulation of MHC class II molecules only in Ii-positive B cells; and 3) these class II molecules reach intracellular compartments where BCR targets exogenous Ag. No differences in expression of adhesion and costimulatory molecules or in the presentation of soluble peptides were detectable between Ii-positive and -negative B cells. Therefore, the BCR delivers its ligand to compartments containing MHC class II-Ii complexes and bypasses the Ii-independent presentation pathway. The linked roles of Ag internalization and B cell activation of the BCR leads to potent Ii-dependent presentation in splenic B cells.

Animals↗

PubMed1999

Dendritic cell presentation of antigens from apoptotic cells in a proinflammatory context: role of opsonizing anti-beta2-glycoprotein I antibodies.

OBJECTIVE: To verify whether opsonization of apoptotic cells skews the outcome of apoptotic cell antigen presentation by dendritic cells (DCs). METHODS: RMA cells, which were engineered with a mutant ovalbumin (OVA) protein and were devoid of the leader secretory sequence (OVA-RMA), underwent ultraviolet irradiation to induce apoptosis. Binding of anti-beta2-glycoprotein I antibodies (anti-beta2GPI) and phagocytosis of apoptotic cells were assessed by flow cytometry and confocal microscopy. Presentation of processing antigens and major histocompatibility complex (MHC) class II-restricted or MHC class I-restricted antigens was assessed using OVA-specific T cell hybridomas. RESULTS: Anti-beta2GPI facilitated presentation of epitopes from internalized apoptotic cells to MHC class II-restricted, but not to class I-restricted, T lymphocytes. DCs challenged with supernatants of apoptotic cells did not activate OVA-specific T cells, making it unlikely that anti-beta2GPI complexed with antigen released from dying cells plays a role in antigen presentation. DCs challenged with low numbers of anti-beta2GPI-opsonized apoptotic cells secreted interleukin-1beta (IL-1beta), tumor necrosis factor alpha, and IL-10 in an autocrine/paracrine manner. CONCLUSION: Opsonization influences the outcome of the disposal of low numbers of apoptotic cells by DCs. This implies that soluble factors bound to apoptotic cells modulate their immunogenicity.

Antibodies↗

PubMed1999

Delayed clearance of apoptotic lymphoma cells allows cross-presentation of intracellular antigens by mature dendritic cells.

Single cells are deleted from the midst of living tissue during normal turnover and embryogenesis. This event is not associated with inflammation or autoimmunity. Little is known of the clearance of apoptotic cells during dangerous situations, accompanied by extensive cell death and tissue damage: when macrophages are overwhelmed by apoptotic cells, other phagocytes, including immature dendritic cells (DCs), may become involved. DCs efficiently present antigens derived from the processing of internalized apoptotic bodies to class I- and class II-restricted T cells. Antigen presentation results either in T cell activation or in their functional blockade. The outcome is influenced by pro-inflammatory maturative signals: efficient T cell cross-priming requires fully mature DCs. Here we discuss in vitro data suggesting that the number of apoptotic cells that die at a given time influences DC maturation and therefore their ability to uptake antigens from apoptotic cells and cross-activate T lymphocytes.

3T3 Cells↗

PubMed1999

Intraductal papillary carcinoma of the parotid gland with low malignancy.

A peculiar case of papillary carcinoma arising in the parotid gland is reported. A 68-year-old woman presented with a right, painless, parotid mass, measuring approximately 3 cm in greatest diameter. A conservative parotidectomy was performed. Histologically, the neoplasm showed exophytic papillary projections into a cavity. The cells were focally suggestive of epidermoidal differentiation, whereas a transitional differentiation was noted in other portions, as in bladder papilloma. Immunohistochemical studies showed strong positivity of the neoplastic cells for cytokeratin and weak positivity for PCNA and Mib-1. We classified this neoplasm among the papillary tumors with a low-grade of malignancy.

Aged↗

PubMed1999

Idiopathic chronic fatigue and chronic fatigue syndrome: a comparison of two case-definitions.

The aim of the study was to compare the signs and symptoms of individuals meeting two different definitions of chronic fatigue syndrome (CFS). Ninety-four patients fitting the eligibility criteria for idiopathic fatigue were enrolled into the study. Of the 94 patients, 48 met the 1988 definition of CFS, 20 the 1994 (but not the 1988) definition of CFS, and 26 met neither definition. The 1994 defined cases were more likely than 1988 defined cases, and non-syndromal individuals to be male, married, and high school educated. The 1994 cases were less likely than 1988 cases to present acute onset, self reported sore throat, mild fever lymphadenopathy, pharyngitis. In conclusion, the 1994 criteria increased the number of patients classified as CFS; however, those who fit only the 1994 criteria were less likely to have an acute symptomatic onset and signs and symptoms suggestive of an infectious process.

Adolescent↗

PubMed1998

Bystander apoptosis triggers dendritic cell maturation and antigen-presenting function.

Physiologic cell death via apoptosis occurs without inflammation or autoimmunity. Here, we investigated the outcome of the interaction of apoptotic cells with dendritic cells (DCs), which are potent professional APCs. DCs internalized apoptotic cells and processed them for presentation to both MHC class I- and class II-restricted T cells with an efficiency that was dependent upon the number of apoptotic cells. The latter event was accompanied by the autocrine/paracrine secretion of IL-1beta and TNF-alpha, with eventual DC maturation. High numbers of apoptotic cells, mimicking a failure of their in vivo clearance, are therefore sufficient to trigger DC maturation and the presentation of intracellular Ags from apoptotic cells, even in the absence of exogenous "danger" signals.

Animals↗

PubMed1998

Dendritic cell maturation and antigen presentation in the absence of invariant chain.

In immature dendritic cells (DCs), major histocompatibility complex class II molecules accumulate in peptide-loading compartments and, during DC maturation, are exported to the cell surface in response to inflammatory stimuli. Moreover, it has recently been proposed that DCs have specific mechanisms of antigen uptake and delivery into major histocompatibility complex class II-loading compartments. B cells bearing a genetically disrupted invariant chain gene (Ii -/-) show alterations in the transport and function of class II molecules. We herein report that DCs derived from Ii -/- H2(k) but not Ii -/- H2(b) mice undergo normal maturation in response to tumor necrosis factor alpha and show a high degree of class II surface expression. Class II molecules are accumulated in cathepsin D- and H2-M-positive compartments in immature Ii -/- DC and, during DC maturation, are exported to the cell membrane as compact dimers. Ii -/- DCs present putative Ii-dependent hen egg lysozyme-derived epitopes to T cells. These data support the existence of Ii-independent molecular requirements for class II transport and peptide loading in DCs.

Animals↗

PubMed1998

Mycobacterium tuberculosis exploits the CD95/CD95 ligand system of gammadelta T cells to cause apoptosis.

Vgamma9/Vdelta2+ T cells specifically recognize Mycobacterium tuberculosis in vitro and are precociously recruited in early mycobacterial lesions. Even if gammadelta T cells are only fortuitously detected in granulomas or bronchoalveolar lavages of patients with active pulmonary tuberculosis, a role in shaping the mature alphabeta T cell response against M. tuberculosis is substantiated. Here we provide a molecular explanation for this paradox: the engagement of the gammadelta TCR by mycobacterial antigens induced the expression of CD95 ligand (CD95L) by chronically activated CD95+/CD95L- gammadelta T lymphocytes. The receptor was functional, as CD95/CD95L interaction triggered the bystander death of CD95+ cells by apoptosis. Cell death was abolished by CD95-blocking antibodies. The transient accumulation at the site of infection of CD95L+ gammadelta lymphocytes, capable of interacting with CD95+ leukocytes attracted by the response towards the pathogen, may determine the characteristics of the ensuing granulomatous disease.

Apoptosis↗

PubMed1998

Apoptotic cell clearance in systemic lupus erythematosus. I. Opsonization by antiphospholipid antibodies.

OBJECTIVE: To verify whether antiphospholipid antibodies (aPL) recognize and opsonize apoptotic human cells. METHODS: Apoptosis was induced via CD95 crosslinking or ultraviolet irradiation. IgG and anti-beta2-glycoprotein I (anti-beta2-GPI) antibodies were purified from patient sera by affinity chromatography. The aPL that bound to apoptotic cells were assessed by flow cytometry, and the subdomains recognized were identified by confocal microscopy. Human macrophages were derived from monocytes, and their ability to phagocytose 3H-labeled apoptotic bodies, whether opsonized or not opsonized by aPL, was assessed. Tumor necrosis factor alpha (TNF alpha) secretion was evaluated by enzyme-linked immunosorbent assay. RESULTS: The aPL, but not control Ig or Ig from aPL-negative patients, bound to apoptotic cells, but not to viable cells. Nuclear antigens were not recognized. Opsonization of apoptotic cells by aPL substantially enhanced recognition and binding by scavenger macrophages, with massive TNF alpha secretion. CONCLUSION: Antiphospholipid antibodies facilitate apoptotic cell clearance by macrophages and trigger TNF alpha release, possibly enhancing the immunogenicity of the autoantigens they contain.

Antibodies, Antiphospholipid↗

PubMed1998

Apoptotic cell clearance in systemic lupus erythematosus. II. Role of beta2-glycoprotein I.

OBJECTIVE: To analyze the contribution of beta2-glycoprotein I (beta2-GPI) to apoptotic cell recognition by antiphospholipid antibodies (aPL) and macrophages from patients with autoimmune disease. METHODS: Phosphatidylserine expression by Jurkat cells undergoing apoptosis upon CD95 crosslinking or ultraviolet irradiation was verified by confocal microscopy of cells stained with fluorescein isothiocyanate-labeled annexin V. Beta2-GPI was purified by heparin/cationic-exchange chromatography and was biotinylated or used to purify beta2-GPI-specific antibodies by affinity chromatography. Binding to apoptotic cells was assessed by flow cytometry. The clearance of 3H-labeled, apoptotic cells by macrophages was assessed by beta counting. RESULTS: The array of epitopes generated by beta2-GPI association with apoptotic cells specifically targets their recognition and is required for their opsonization by human aPL. Nevertheless, beta2-GPI is not required for apoptotic cell clearance by human macrophages in the absence of aPL. CONCLUSION: The proinflammatory clearance of aPL-opsonized apoptotic cells, but not the nonphlogistic clearance of apoptotic cells by scavenger macrophages, depends on beta2-GPI.

Antibodies, Antiphospholipid↗

PubMed1998

Dendritic cells preferentially internalize apoptotic cells opsonized by anti-beta2-glycoprotein I antibodies.

Dendritic cells (DC) are potent antigen-presenting cells involved in the initiation of immune responses, including those directed towards self antigens. Immature DC capture soluble antigens by macropinocytosis or c-type lectin receptor-mediated endocytosis and particulate by phagocytosis, including Fc receptor-mediated phagocytosis. Apoptosis is accompanied by the clustering of intracellular autoantigens, which are also selectively cleaved and phos-phorylated, and by the exposure of anionic phospholipids (phosphatidyl-serine, PS). Anti-phospholipid antibody (aPL) detection correlates with an increased risk of developing autoimmune syndromes. In this study apoptosis was induced by UV irradiation, growth factor deprivation or exposure to protein synthesis inhibitors of murine cells and verified by confocal microscopy and flow cytometry. Apoptotic cells were recognized by a panel of anti-beta2-glycoprotein I (beta2-GPI) aPL monoclonal antibodies, but not by isotype-matched antibodies. The binding restricted to membrane domains, corresponding to apoptotic blebs, was not affected by the stimulus initiating apoptosis and was specific, since it required the association of the beta2-GPI co-factor to the apoptotic membrane. aPL-binding successfully transformed apoptotic cells in an efficient phagocytic substrate for murine immature DC, possibly skewing their immunogenicity in vivo.

Animals↗

PubMed1998

Heterogeneity and susceptibility to apoptosis of human renal carcinoma cells in vitro.

Tumors are heterogeneous in terms of morphology and susceptibility to drugs or radiation. Among primary and metastatic cells of a human renal carcinoma, a population (type II) of larger cells with prominent nucleoli, eosinophilic globules of intermediate filaments in paranuclear bundles, margination of subcellular organelles and peripheral pools of glycogen was evident. Paranuclear structures were recognized by monoclonal antibodies specific for cytokeratin 8, 18 and 19, but not by vimentin specific antibodies. We propagated a cell line in vitro (referred to as BKR cells), and observed culture in vitro, the almost complete disappearance of the type II cells. Pharmacological agents that influence cell differentiation, such as retinoic acid, rescued the expression of type II cells in vitro. Long-term treatments with insulin or alpha-interferon, but not with the epithelial growth factor (EGF), similarly differentiated BKR cells and abated their susceptibility to spontaneous and actinomycin-D induced apoptosis. These data support the contention that differentiation of tumor cells is actively maintained in vivo and further strengthen the caveat on tumor lines stabilized in vitro, that poorly represent the morphologic and antigenic heterogeneity of neoplasms in vivo.

Apoptosis↗

PubMed1998

AIDS-related non-Hodgkin's lymphomas from an Italian area.

In a retrospective study, 42 (7.7%) of 545 patients with AIDS from a single area of Italy had non-Hodgkin's lymphoma (28 systemic and 14 primary central nervous system lymphomas). The improved outcome and survival of treated patients outlines the clinical benefit of antineoplastic treatment in selected cases.

Antineoplastic Combined Chemotherapy Protocols↗