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Biomedical subjects

Santo Landolfo

Publications and source records attributed to Santo Landolfo.

At least 19 recordsLinked to original sources
PubMed2006

Alpha- and betapapillomavirus E6/E7 genes differentially modulate pro-inflammatory gene expression.

Keratinocytes, the target cell of human papillomavirus (HPV) infection, can produce numerous cytokines and pro-inflammatory molecules which are important for the generation of an effective immune response. How this biological response, which involves the tumor stroma, is affected by the HPV oncoproteins within the epithelial cell itself is not clear. Here it is shown that oncoproteins of different HPV genotypes (alpha- versus beta-HPV genus) alter the expression of pro-inflammatory molecules in early passage primary human keratinocytes and the immortalized cell line HaCaT. HPV5 E6/E7 oncoproteins significantly induced interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1) expression. By contrast, the same molecules were down-regulated or not modulated in HPV16 E6/E7 transduced keratinocytes. Interestingly, HPV38 oncoproteins expression resulted in a lower induction of pro-inflammatory molecules, resembling the behavior displayed by the mucosal carcinogenic HPV16. Finally, inducible nitric oxide synthase (iNOS) expression levels and nitric oxide (NO) production were induced at similar levels by all the HPV genotypes tested. These results further emphasize the different biological activities among HPV genotypes, and offer new insights into HPV-associated skin diseases.

Alphapapillomavirus↗
PubMed2006

Targeting the NF-kappaB pathway through pharmacological inhibition of IKK2 prevents human cytomegalovirus replication and virus-induced inflammatory response in infected endothelial cells.

Endothelial cells are important reservoirs for human cytomegalovirus (HCMV) replication, dissemination and persistence. HCMV infection of endothelial cells has been associated with a proinflammatory response characterized by an increased expression of chemokines and adhesion molecules and modulation of angiogenesis. Many of the host proinflammatory genes augmented in HCMV-infected endothelial cells are regulated, at least in part, by the NF-kappaB pathway. HCMV is a potent activator of NF-kappaB through the IKK-IkappaB signaling axis. To explore whether inhibition of HCMV-induced NF-kappaB activation may interfere with the onset of virus-associated inflammatory response, we measured the effects of the specific IKK2 inhibitor AS602868 on the expression of a panel of proinflammatory genes in HUVEC cells infected with a clinical isolate. Treatment of infected HUVEC with AS602868 was shown to impair HCMV-induced NF-kappaB activity, IE gene expression, viral replication and to prevent HCMV-induced upregulation of ICAM-1, IL-8, RANTES, IP-10, I-TAC and COX-2 gene expression. Consistent with these results, HCMV-mediated upregulation of another NF-kappaB-dependent gene, the plasminogen inhibitor type-1, a regulatory factor of endothelial proliferation and angiogenesis, was abrogated by AS602868. These results suggest that inhibition of HCMV-induced IKK-NF-kappaB activation may be of interest to limit the virus-induced inflammatory response of infected endothelial cells.

Chemokine CCL5↗
PubMed2006

Effect of high-risk human papillomavirus oncoproteins on p53R2 gene expression after DNA damage.

The p53R2 protein is a p53-inducible small subunit of ribonucleotide reductase. It plays a crucial role in p53-dependent cellular response to DNA damage and oxidative stress by providing deoxyribonucleotides (dNTPs) to the DNA repair machinery and by scavenging reactive oxygen species (ROS). To investigate the effects of high-risk human papillomavirus (HPV) oncoproteins on p53R2 expression after DNA damage, we analyzed the p53R2 protein levels in human cells ectopically expressing the HPV-16 E6 and E7 genes, and in the HPV-positive cancer cell lines SiHa, CaSki and HeLa, exposed to adriamycin or to H(2)O(2). We found that in normal cells, p53R2 expression is efficiently induced by both H(2)O(2) and adriamycin, supporting the role of p53R2 in cellular response to oxidative stress. Ectopic expression of E6 impaired p53 and p53R2 induction after DNA damage in human fibroblasts. Moreover, SiHa, CaSki and HeLa cells were unresponsive to H(2)O(2) exposure, and adriamycin induced p53R2 levels only in SiHa cells. Our results imply that high-risk HPV infection may suppress the p53R2-dependent dNTPs supply to the DNA repair system and the ROS scavenging activity; they also suggest that an altered p53R2 response to genotoxins and to oxidative stress may contribute to HPV-induced genetic instability and carcinogenesis.

Blotting, Western↗
PubMed2006

The expression of p16INK4a tumor suppressor is upregulated by human cytomegalovirus infection and required for optimal viral replication.

The human cytomegalovirus (HCMV) induces a replicative senescence program after arresting host cell cycle progression so as to create a favorable environment for its replication. Here, we report that HCMV infection stimulates the expression of p16(INK4a), a direct effector of the senescence phenotype. The increase in p16(INK4a) gene expression was due to an increase in gene transcription, since the expression of a reporter gene driven by the p16(INK4a)-encoding CDKN2A gene promoter was strongly induced by HCMV infection. The results of deletion and mutational analysis of the CDKN2A promoter further suggest the involvement of Ets transcription factors in HCMV-mediated stimulation of p16(INK4a) gene expression. The significance of p16(INK4a) upregulation during the HCMV replicative cycle is underscored by the finding that virus replication was severely impaired in fibroblasts homozygous for an intragenic deletion in CDKN2A locus and devoid of functional p16(INK4a). Moreover, a retrovirus-mediated p16(INK4a) small interfering RNA (p16-siRNA) effectively reduced viral replication, thus providing direct evidence that p16(INK4a) upregulation plays a positive role for HCMV replication.

Artificial Gene Fusion↗
PubMed2006

A novel autoantigen to differentiate limited cutaneous systemic sclerosis from diffuse cutaneous systemic sclerosis: the interferon-inducible gene IFI16.

OBJECTIVE: To investigate the presence and clinical significance of autoantibodies against the interferon-inducible gene IFI16 in systemic sclerosis (SSc), systemic lupus erythematosus (SLE), and other autoimmune diseases. METHODS: Immunohistochemical analysis was used to evaluate the expression of IFI16 in skin biopsy specimens obtained from patients with SSc and patients with SLE. Levels of antibodies against IFI16 in sera from 82 patients with SSc and 100 patients with SLE were determined by enzyme-linked immunosorbent assay. Other autoimmune diseases such as primary Sjögren's syndrome (SS), rheumatoid arthritis (RA), chronic urticaria, and hepatitis C virus (HCV) infection were also examined. RESULTS: Expression of IFI16 was greatly increased and was ubiquitous in all layers of the epidermis and in the dermal inflammatory infiltrates of lesional skin from both patients with SLE and patients with SSc. Patients with SLE, those with primary SS, and those with SSc exhibited significantly higher anti-IFI16 IgG antibody levels compared with normal controls (for SLE, P < 0.002; for primary SS, P < 0.001; for SSc, P < 0.0005). Anti-IFI16 titers above the ninety-fifth percentile for control subjects were observed in 26% of the patients with SLE, 50% of those with primary SS, and 21% of those with SSc (28% of patients with limited cutaneous SSc [lcSSc] versus 4% of patients with diffuse cutaneous SSc [dcSSc]). In contrast, the prevalence of anti-IFI16 was 4% in patients with RA, 5% in those with chronic urticaria, and 13% in those with HCV infection. CONCLUSION: The results of this study provide evidence that an IFN-inducible gene, IFI16, may be involved in the pathophysiologic mechanisms of connective tissue disorders such as SSc. Moreover, a strict correlation with lcSSc was also demonstrated, thus providing a novel tool in the differential diagnosis of lcSSc from dcSSc.

Adult↗
PubMed2006

Head and neck squamous cell carcinoma: role of the human papillomavirus in tumour progression.

High risk human papilloma viruses (HPVs) have been shown to be independent risk factors for anogenital tract cancers, and have also been detected in head and neck squamous cell carcinomas (HNSCC). The aim of our study was to determine the prevalence of HPV DNA in a group of 47 squamous cell carcinomas of the oropharynx and the oral cavity, and to compare the clinical behaviour of HPV positive and negative tumours. We also assessed the proliferation index, as evaluated by Ki67 immunohistochemistry positivity, and the level of p53 reactivity. HPV DNA was found in 50% of carcinomas of the oropharynx and 36% in those of the oral cavity, the only genotype detected being HPV 16. Patients with HPV-positive carcinomas had a better overall survival than those with HPV-negative carcinomas. Our data suggest that HPV-positive oropharyngeal cancers comprise a distinct disease entity with an improved prognosis.

Adult↗
PubMed2005

A cell-based high-throughput assay for screening inhibitors of human papillomavirus-16 long control region activity.

Cervical carcinomas express human papillomavirus (HPV) E6 and E7 oncoproteins, which are required to maintain the proliferative state of cancer cells. Repression of E6 and E7 expression results in acquisition of senescent phenotype and in the rescue of functional p53 and p105(Rb) pathways; therefore, therapies directed against either viral protein may be beneficial. However, the systems to study HPV in vitro are technically difficult and not convenient for screening of antiviral compounds. This has hampered the discovery of drugs against HPV. Here we describe the generation and use of a high-throughput screening system based on keratinocytes stably transfected with a reporter construct containing the regulatory sequence of E6 and E7 gene transcription (LCR) that allows easy detection of inhibitors of E6 and E7 expression in libraries of synthetic or biological compounds. The assay was used to screen a wide panel of cytokines: among them, IL-4, IL-13, TGF-beta1, TGF-beta2, TGF-beta3, TNF-alpha, IFN-alpha, and IFN-beta were found to induce a strong inhibition of the LCR activity. Our assay provides a validated tool in the search for drugs against HPV-associated cervical carcinomas and allowed the first systematic analysis of the effect of cytokines on the HPV-16 LCR transcriptional activity.

Cell Line↗
PubMed2005

Up-regulation of the interferon-inducible IFI16 gene by oxidative stress triggers p53 transcriptional activity in endothelial cells.

Reactive oxygen species (ROS), including hydrogen peroxide (H2O2), induces injury of endothelium in a variety of pathophysiological conditions, such as inflammation, aging, and cancer. In our study, we characterized the signaling pathway linking oxidative stress induced by sublethal concentrations of H2O2 to p53 in primary human endothelial cells through the interferon (IFN)-inducible gene IFI16. Induction of IFI16 by H2O2 was concentration- and time-dependent (maximum at 50 microM, 6 h after treatment) and down-regulated by pretreatment with N-acetyl-L-cysteine, which acts as an antioxidant. This pathway is a general response to ROS and not specific to H2O2 treatment, as two other ROS-generating compounds, i.e., S-nitroso-N-acetylpenicillamine and tert-butyl hydroperoxide, were equally capable to induce IFI16. Moreover, IFI16 up-regulation is a result of protein accumulation, as expression of corresponding mRNA, assessed by real-time polymerase chain reaction, was not affected. To investigate the mechanism of IFI16 accumulation, cells were incubated for 6 h in the presence of H2O2 or IFN-beta, and then cycloheximide was added to inhibit further protein synthesis. The half-life of IFI16 protein was found to be significantly increased in H2O2-treated cells compared with IFN-beta-treated cells (t1/2 = 120 min vs. > 30 min in H2O2- vs. IFN-beta-treated cells, respectively). An increase of IFI16 was accompanied by interaction with p53 phosphorylated at its N terminus, as shown by immunoprecipitation experiments. Moreover, binding to IFI16 resulted in its transcriptional activation as shown by an increase in the activity of a reporter gene driven by p53-responsive sequences derived from the p21(WAF1) promoter, along with an increase in the p21 mRNA and protein levels. Altogether, these results demonstrate a novel role of IFI16 in the signal transduction pathway that leads to p53 activation by oxidative stress in endothelial cells.

Cell Cycle Proteins↗
PubMed2004

Evidence that the human cytomegalovirus 46-kDa UL72 protein is not an active dUTPase but a late protein dispensable for replication in fibroblasts.

The Human Cytomegalovirus (HCMV) UL72 gene is considered to be the equivalent of the dUTPase gene of the Alpha- and Gamma-herpesviruses. To characterize its function, the expression profiles of UL72 at both the RNA and the protein level were determined. The gene is expressed with a late kinetics and the corresponding UL72 46-kDa protein accumulates late during infection in the cytoplasm of infected cells. The pUL72 was expressed in E. coli and the purified recombinant protein did not display a detectable dUTPase activity. The viral yields of reconstituted HCMV RVDeltaUL72 viruses carrying a deletion within the UL72 ORF demonstrated a moderate growth defect following low MOI infections, whereas their DNA synthesis profiles were not significantly different from those of the parental HCMV RVAD169. These results demonstrate that the UL72 gene product is not a dUTPase and is not essential for replication in human fibroblasts.

Amino Acid Sequence↗
PubMed2004

The interferon-inducible IFI16 gene inhibits tube morphogenesis and proliferation of primary, but not HPV16 E6/E7-immortalized human endothelial cells.

Immunohistochemical analysis has demonstrated that the human IFI16 gene, in addition to the hematopoietic tissues, is highly expressed in endothelial cells and squamous stratified epithelia. In this study, we have developed a reliable HSV-derived replication-defective vector (TO-IFI16) to efficiently transduce IFI16 into primary human umbilical vein endothelial cells (HUVEC), which are usually poorly transfectable. HUVEC infection with TO-IFI16 virus suppressed endothelial migration, invasion and formation of capillary-like structures in vitro. In parallel, sustained IFI16 expression inhibited HUVEC cell cycle progression, accompanied by significant induction of p53, p21, and hypophosphorylated pRb. Further support for the involvement of these pathways in IFI16 activity came from the finding that infection with TO-IFI16 virus does not impair the in vitro angiogenic activity and cell cycle progression of HUVEC immortalized by HPV16 E6/E7 oncogenes, which are known to inactivate both p53 and pRb systems. This use of a reliable viral system for gene delivery into primary human endothelial cells assigns a potent angiostatic activity to an IFN-inducible gene, namely IFI16, and thus throws further light on antiangiogenic therapy employing IFNs.

Angiogenesis Inhibitors↗
PubMed2004

Human cytomegalovirus stimulates cellular IKK2 activity and requires the enzyme for productive replication.

Human cytomegalovirus (HCMV) exploits the host transcription factor NF-kappaB to enhance its own replication, dissemination, and reactivation from latency. Here we report that HCMV infection activates the upstream IkappaB kinase (IKK) complex and that its catalytic IKK2 subunit is required for HCMV-induced NF-kappaB activation, as well as the replication of different HCMV strains. These results indicate that IKK2 is essential for HCMV replication and emphasize the feasibility of blocking NF-kappaB activation as a way of inhibiting infection.

Cell Line↗
PubMed2004

The ribonucleotide reductase R1 homolog of murine cytomegalovirus is not a functional enzyme subunit but is required for pathogenesis.

Ribonucleotide reductase (RNR) is the key enzyme in the biosynthesis of deoxyribonucleotides. Alpha- and gammaherpesviruses express a functional enzyme, since they code for both the R1 and the R2 subunits. By contrast, betaherpesviruses contain an open reading frame (ORF) with homology to R1, but an ORF for R2 is absent, suggesting that they do not express a functional RNR. The M45 protein of murine cytomegalovirus (MCMV) exhibits the sequence features of a class Ia RNR R1 subunit but lacks certain amino acid residues believed to be critical for enzymatic function. It starts to be expressed independently upon the onset of viral DNA synthesis at 12 h after infection and accumulates at later times in the cytoplasm of the infected cells. Moreover, it is associated with the virion particle. To investigate direct involvement of the virally encoded R1 subunit in ribonucleotide reduction, recombinant M45 was tested in enzyme activity assays together with cellular R1 and R2. The results indicate that M45 neither is a functional equivalent of an R1 subunit nor affects the activity or the allosteric control of the mouse enzyme. To replicate in quiescent cells, MCMV induces the expression and activity of the cellular RNR. Mutant viruses in which the M45 gene has been inactivated are avirulent in immunodeficient SCID mice and fail to replicate in their target organs. These results suggest that M45 has evolved a new function that is indispensable for virus replication and pathogenesis in vivo.

Animals↗
PubMed2003

The human cytomegalovirus.

Human cytomegalovirus (HCMV), a betaherpesvirus, represents the major infectious cause of birth defects, as well as an important pathogen for immunocompromised individuals. The viral nucleocapsid containing a linear double-stranded DNA of 230 kb is surrounded by a proteinaceous tegument, which is itself enclosed by a loosely applied lipid bilayer. Expression of the HCMV genome is controlled by a cascade of transcriptional events that leads to the synthesis of three categories of viral proteins designated as immediate-early, early, and late. Clinical manifestations can be seen following primary infection, reinfection, or reactivation. About 10% of infants are infected by the age of 6 months following transmission from their mothers via the placenta, during delivery, or by breastfeeding. HCMV is a significant post-allograft pathogen and contributes to graft loss independently from graft rejection. Histopathologic examination of necropsy tissues demonstrates that the virus enters via the epithelium of the upper alimentary, respiratory, or genitourinary tracts. Hematogenous spreading is typically followed by infection of ductal epithelial cells. Infections are kept under control by the immune system. However, total HCMV clearance is rarely achieved, and the viral genome remains at selected sites in a latent state. Virological and molecular detection of HCMV, as well as serological demonstration of a specific immune response, are used for diagnosis. Treatment of HCMV infections is difficult because there are few options. The presently available drugs produced a significant clinical improvement, but suffer from poor oral bioavailability, low potency, development of resistance in clinical practice, and dose-limiting toxicities.

Antiviral Agents↗
PubMed2003

Human cytomegalovirus requires cellular deoxycytidylate deaminase for replication in quiescent cells.

We have previously observed that the expression of two thymidylate biosynthesis enzymes, dihydrofolate reductase and thymidylate synthase (TS), is upregulated in quiescent human fibroblasts infected with human cytomegalovirus (HCMV). Here, we have demonstrated that HCMV increases expression of the cellular deoxycytidylate deaminase (dCMP deaminase), which provides the substrate for TS by converting dCMP to dUMP. We observed an increase in dCMP deaminase protein levels, whereas deoxyuridine triphosphatase (dUTPase), another cellular enzyme that may provide dUMP by hydrolysing dUTP, was undetectable. The essential requirement of cellular dCMP deaminase for productive HCMV replication was further emphasized by showing that a precursor of a potent dCMP deaminase inhibitor, zebularine, suppressed virus replication and DNA synthesis. These results suggest that HCMV exploits the host's dCMP deaminase activity to replicate in quiescent cells.

Cells, Cultured↗
PubMed2002

The interferon-inducible gene, Ifi204, acquires malignant transformation capability upon mutation at the Rb-binding sites.

p204 overexpression in retinoblastoma (Rb)-/- mouse embryo fibroblasts or transfection of p204 mutated at both Rb-binding sites confer growth advantages, resulting in a significantly higher number of foci in a cell focus assay. To investigate the possibility that mutated p204 acquires malignant transformation capability, NIH3T3 cells were stably transfected with the expression vector pRcRSV204 double-mutant (p204dm) harboring both the C-terminal deletion up to amino acid 568 and the point mutation from glutamic acid to lysine at position 427, and analyzed for markers typical of cell immortalization and transformation. We detected a greater abundance of cell colonies in soft agar with p204dm-expressing cells than vector control cells. The p204dm-transfected cells also displayed two other characteristics associated with malignant transformation, i.e. growth under low-serum conditions and formation of tumors in athymic nude mice. Moreover, their telomerase activity was significantly higher than in the vector control cells. It would thus seem that p204, devoid of functional Rb-binding motifs, can become oncogenic.

3T3 Cells↗
PubMed2002

Cloning and characterization of two catechol 1,2-dioxygenase genes from Acinetobacter radioresistens S13.

Two novel catechol 1,2-dioxygenase (C 1,2-O) genes have been isolated from an Acinetobacter radioresistens strain that grows on phenol or benzoate as sole carbon and energy source. Designated as catA(A) and catA(B), they encode proteins composed of 314 and 306 amino acids, whose deduced sequences indicate that they have approximately 53% identity, whereas their NH2-terminal and COOH-terminal regions have no sequences in common. This may explain their different thermal and pH stability. Polyclonal antibodies raised against an amino-terminal CatA(A) peptide or the whole CatA(B) protein were used to establish their inducible and differential expression patterns upon bacterial growth in phenol or benzoate. The CatA(A) protein (IsoA) was induced by both phenol and benzoate though with different kinetics, whereas the catA(B) product (IsoB) was constitutively produced at low levels that increased only during growth in the presence of benzoate.

Acinetobacter↗
PubMed2002

The interferon system: an overview.

Interferons are a family of related and naturally occurring signal proteins grouped in three major species (alpha, beta and gamma) according to their cellular origin and inducing agents and historically described for their antiviral activity. Upon binding to specific receptors they lead to the activation of a signal transduction pathway that activates a broad range of genes, that are now known involved not only in antiviral but also in immunomodulatory and antiproliferative activities. The inhibition of virus growth and/or cell proliferation by interferons is associated with several physiological changes, some of which depend on the activity of specific proteins that are interferon-inducible. This review will attempt to illustrate the history and main properties of the interferon system, taking a look at recent discoveries about some interferon-inducible genes.

Animals↗