Biomedical subjects

M Gariglio

Publications and source records attributed to M Gariglio.

At least 19 recordsLinked to original sources

PubMed2004

Altered patterns of the interferon-inducible gene IFI16 expression in head and neck squamous cell carcinoma: immunohistochemical study including correlation with retinoblastoma protein, human papillomavirus infection and proliferation index.

AIMS: To investigate whether the expression of interferon (IFN)-inducible gene IFI16 is inversely related to proliferative activity in vivo, we compared immunohistochemical reactivity of IFI16 in a series of head and neck squamous cell carcinomas (HNSCCs) with their proliferation index and the cell cycle regulator pRb. As human papillomavirus (HPV) infection is manifested by changes in the function or expression level of host genes such as IFN-inducible genes, we also investigated the presence of HPV DNA to determine whether head and neck cancers associated with HPV DNA can be distinguished from tumours that are presumably transformed by other mechanisms. METHODS: Thirty-six HNSCCs were evaluated for IFI16, pRb and Ki67 expression by immunohistochemistry. The presence of HPV was also detected by polymerase chain reaction. Nine tumours were located in the oropharynx (tonsillar area) and 27 in the larynx. RESULTS: HPV DNA was found in 14 of 25 (56%) laryngeal SCCs and in five of nine (56%) tonsillar SCC specimens examined; 17 out of the 19 HPV-DNA-positive cases showed high-grade IFI16 expression. Overall, proliferative activity was significantly related to tumour differentiation and histological grading. IFI16 protein expression was significantly inversely correlated with Ki67 (P = 0.039). Low-proliferating tumours positive for IFI16 staining showed a marked expression of pRb and a better prognosis than those whose tumours had low IFI16, pRb levels and a high proliferation index. CONCLUSIONS: To our knowledge, this is the first expression analysis of the IFN-inducible IFI16 gene in HNSCC. Low-proliferating tumours positive for IFI16 staining showed a marked expression of pRb and a better prognosis than those whose tumours had low IFI16, pRb levels and a high proliferation index.

Aged↗

PubMed2004

Prevalence of autoantibodies against structure specific recognition protein 1 in systemic lupus erythematosus.

Antibodies (Abs) against the structure specific recognition protein 1 (SSRP1) were reported in a small systemic lupus erythematosus (SLE) series but not in other systemic autoimmune diseases. The aim of the study was to confirm the selective presence of anti-SSRP1 Abs in a larger SLE series and to evaluate their relationship with disease activity and other immune markers. Anti-SSRP1 Abs were investigated by a 'home made' ELISA in: 120 SLE, 65 rheumatoid arthritis (RA), 51 systemic sclerosis (SSc), 23 Churg-Strauss syndrome (CSS) and 40 idiopathic autoimmune urticaria (IAU) patients and 190 healthy controls. Sera from MRL lpr/lpr and Balb-c mice were also tested. Anti-SSRP1 Abs were detected in 43 SLE (35.8%), nine SSc (17.6%), eight RA (12.3%), six IAU (15%), three CSS (13%) patients and five healthy controls (2.6%). Antibody prevalence and titers were significantly higher in SLE patients than in sera from both normal and disease controls. Anti-SSRP1 Ab activity was also detected in sera from MRL lpr/lpr but not Balb-c mice. The antibodies did not correlate with the disease activity evaluated as the ECLAM index score and were more prevalent in patients without renal involvement. No correlation was found with other serum autoantibodies. Our results confirm that anti-SSRP1 Abs are associated with but not specific for the lupus disease.

Adolescent↗

PubMed2001

The interferon-inducible 204 gene is transcriptionally activated by mouse cytomegalovirus and is required for its replication.

Infection of cells with viable or UV-inactivated murine cytomegalovirus (MCMV) increased the IFN-inducible 204 gene at both the mRNA and the protein levels. The activity of a reporter gene driven by the mouse Ifi204 promoter induced following virus infection showed that this increase was due to transcriptional activation. Moreover, FACS analysis of infected mouse embryo fibroblasts (MEF) stably transfected with a p204-dominant-negative mutant (p204dmMEF) revealed that they do not accumulate at the G1/S border in the same way as infected MEF transfected with the empty vector (neoMEF). MCMV DNA synthesis is significantly delayed (144 h in p204dmMEF vs 72 h in neoMEF), due to retarded expression of viral genes, namely, IE1 and DNA polymerase, as shown by Western blot comparison of p204dmMEF and neoMEF extracts. These results demonstrate that MCMV may exploit the Ifi204 gene to regulate the cell cycle and enhance its DNA synthesis.

Animals↗

PubMed2001

Control of murine cytomegalovirus replication in salivary glands during acute infection is independent of the Fas ligand/Fas system.

The course of mouse cytomegalovirus (MCMV) infection was compared between mutant C57BL/6 (B6) mice deficient in either perforin (perf-/-), or perforin, granzyme A and B (perfxgzmAxB-/-), and B6 gld mice lacking functionally active Fas ligand to elucidate the contribution of the two main cytolytic pathways in the early control of MCMV infection. At 15 and 30 days post infection (p.i.) virus titers were elevated in salivary glands of perf-/- and perfxgzmAxB-/-, but almost undetectable in those of mutant gld and C57BL/6 wild-type mice. No virus was detectable in lung and spleen tissues of the mutant or B6 mice at the time points tested. At 15 days p.i., scanty lymphocytic periductal infiltration was seen in salivary glands of perf-/- and perfxgzmAxB-/; these pathological alterations were minimal at 30 days p.i.. In contrast, no pathological alterations were seen in the respective organs of infected B6 and gld mice at the two time points p.i.. At 15 days p.i., reactive follicles were observed in the white pulp of spleen tissues from both mutant and B6 mice, but at 30 days p.i. only in those of mutant mice. No inflammatory responses were seen in the lung tissues of any of the four mouse strains tested. Together with previous observations (Riera et al.. 2000), the results demonstrate that both perforin and granzymes A/B, but not the FasL/Fas system are critical for viral elimination in salivary glands during the acute phase of infection. However, for the long-term control of MCMV infection, neither of the two cytolytic pathways seem to be necessary.

Acute Disease↗

PubMed2000

The retinoblastoma protein is an essential mediator that links the interferon-inducible 204 gene to cell-cycle regulation.

We have previously demonstrated that overexpression of p204, a member of the Ifi 200 gene family, inhibits growth, delays G0/G1 progression into S phase, and impairs E2F-mediated transcriptional activity. In this study, we show that p204 directly binds the retinoblastoma protein (pRb) in vivo to exert its activity. Transient p204 overexpression in Rb+/+ mouse embryo fibroblasts (MEF) inhibits cell proliferation, but does not affect cell growth in MEF derived from Rb-/- mice. Two human cell lines, Saos2 and C33A, bearing an inactive pRb, but not primary human embryo fibroblasts, are resistant to the p204 antiproliferative activity. p204 contains two 200 amino acid motifs, designated as type a or b domains, each containing a canonical Rb binding motif (LXCXE). When dominant-negative mutants at the Rb binding motif were transfected in Rb+/+ MEF, p204 lost its ability to inhibit cell growth, delay cell transition from G1 to S phase, and impair DNA synthesis. Moreover p204 overexpression in Rb+/+ MEF led to a significant decrease of both DHFR and PCNA proteins, two S phase markers. By contrast, this effect was not observed when Rb+/+ MEF were transfected with a p204 mutated at both Rb binding sites. Finally, overexpression of the LXCXE p204 mutant rendered Rb+/+ MEF resistant to the IFN-alpha antiproliferative activity, in comparison to the untransfected Rb+/+ MEF. As expected, Rb-/- cells were unsensitive to the IFN-alpha induced growth inhibition. Taken as a whole, these results suggest that (i) p204 contributes to the IFN-alpha antiproliferative activity and (ii) the primary target of p204 leading to efficient G1 arrest as well as to blockade of DNA replication from G1 phase is the pRb regulatory system.

3T3 Cells↗

PubMed2000

Murine cytomegalovirus replication in salivary glands is controlled by both perforin and granzymes during acute infection.

The course of mouse cytomegalovirus (MCMV) infection was compared between wild-type and mutant C57BL / 6 (B6) mice deficient in either RAG-2, perforin, granzyme A, granzyme B or combinations thereof at two time points post infection (p. i.). At day 15 p. i., virus titers were similarly elevated in salivary glands of all mutant, but not wild-type B6 mice and undetectable in lung and spleen tissues of any of the mouse strains. Significant pathological alterations were only seen in salivary glands and spleen from RAG2(- / -), but not in those from other mice whereas few inflammatory foci were observed in lung tissues of all mice except B6. At day 30 p. i., elevated virus titers were observed only in salivary glands, lung and spleen from RAG2(- / -), but in none of the other mice, and were accompanied by extended pathological alterations in all three organs. The data extend previous reports on the critical role of NK / CD8(+) T cells in the early control of MCMV infection by showing that both perforin and granzymes A / B contribute to viral elimination in salivary glands; however, neither of the three molecules alone seem to be indispensable for the final control of infection.

Animals↗

PubMed2000

Chronic myeloid leukemia cells resistant to interferon-alpha lack STAT1 expression.

INTRODUCTION: Interferon-alpha (IFN) plays a role in the management of different neoplasias, particularly those of hematological origin. The mechanisms of action of IFN are still poorly understood and the individual response is unpredictable. In the present study, the pattern of intracellular gene expression following in vitro and in vivo exposure of chronic myeloid leukemia (CML) cells to IFN was evaluated and correlated with the response to in vivo treatment with IFN. MATERIALS AND METHODS: CML patients in different phases of the disease were studied. The pattern of expression of two IFN-inducible proteins involved in IFN-mediated biological activities, the p91 and p84 proteins (STAT1alpha and STAT1beta), components of the IFN-stimulated gene factor 3 (ISGF3) complex and the enzyme 2'-5' oligoadenylate synthetase (2'-5' OASE) were investigated by Western blot in peripheral blood mononuclear cells stimulated or not in vitro by IFN. RESULTS AND CONCLUSIONS: In 6/9 patients evaluated before starting treatment, STAT1 was expressed either constitutively or after in vitro stimulation by IFN. In three cases, STAT1 remained negative even after in vitro activation. The pattern of protein expression correlated with the subsequent hematological response to prolonged in vivo IFN administration: the presence of STAT1 being associated with the clinical response to IFN and the absence and non-inducibility of STAT1 with resistance to IFN. This was further substantiated by studies carried out in ten patients analyzed at the time of a documented clinico-hematological response or resistance to the in vivo administration of IFN. Finally, in order to establish whether the pattern of response to IFN treatment could be predicted at diagnosis, cells cyropreserved at diagnosis from patients with a documented complete response, confirmed also by cytogenetic negativity, or resistance, were studied. While complete responders proved STAT1 positive, none of the four resistant cases ever expressed STAT1. The expression of 2'-5' OASE did not correlate with the clinical response to IFN. This study documents the pivotal role of STAT1 in the in vitro and in vivo responses of CML cells to IFN. The constitutive or induced presence or absence of STAT1 shows a predictive correlation with the response or resistance to treatment with IFN and could be utilized to identify, at diagnosis, resistant patients who may be spared an expensive and unnecessary prolonged IFN administration.

Adult↗

PubMed2000

Murine cytomegalovirus stimulates cellular thymidylate synthase gene expression in quiescent cells and requires the enzyme for replication.

Herpesviruses accomplish DNA replication either by expressing their own deoxyribonucleotide biosynthetic genes or by stimulating the expression of the corresponding cellular genes. Cytomegalovirus (CMV) has adopted the latter strategy to allow efficient replication in quiescent cells. In the present report, we show that murine CMV (MCMV) infection of quiescent fibroblasts induces both mRNA and protein corresponding to the cellular thymidylate synthase (TS) gene, which encodes the enzyme that catalyzes the de novo synthesis of thymidylic acid. The increase in TS gene expression was due to an increase in gene transcription, since the activity of a reporter gene driven by the mouse TS promoter was induced following MCMV infection. Mutagenesis of the potential E2F-responsive element immediately upstream from the TS essential promoter region abolished the virus-mediated stimulation of the TS promoter, suggesting that the transactivating activity of MCMV infection was E2F dependent. Cotransfection experiments revealed that expression of the viral immediate-early 1 protein was sufficient to mediate the increase in TS promoter activity. Finally, MCMV replication and viral DNA synthesis were found to be inhibited by ZD1694, a quinazoline-based folate analog that inhibits TS activity. These results demonstrate that upregulation of cellular TS expression is required for efficient MCMV replication in quiescent cells.

3T3 Cells↗

PubMed2000

Expression of an altered ribonucleotide reductase activity associated with the replication of murine cytomegalovirus in quiescent fibroblasts.

Ribonucleotide reductase (RNR) is an essential enzyme for the de novo synthesis of both cellular and viral DNA and catalyzes the conversion of ribonucleoside diphosphates into the corresponding deoxyribonucleoside diphosphates. The enzyme consists of two nonidentical subunits, termed R1 and R2, whose expression is very low in resting cells and maximal in S-phase cells. Here we show that murine cytomegalovirus (MCMV) replication depends on ribonucleotide reduction since it is prevented by the RNR inhibitor hydroxyurea. MCMV infection of quiescent fibroblasts markedly induces both mRNA and protein corresponding to the cellular R2 subunit, whereas expression of the cellular R1 subunit does not appear to be up-regulated. The increase in R2 gene expression is due to an increase in gene transcription, since the activity of a reporter gene driven by the mouse R2 promoter is induced following virus infection. Cotransfection experiments revealed that expression of the viral immediate-early 1 protein was sufficient to mediate the increase in R2 promoter activity. It was found that the viral gene M45, encoding a putative homologue of the R1 subunit, is expressed 24 and 48 h after infection. Meanwhile, we observed an expansion of the deoxyribonucleoside triphosphate pool between 24 and 48 h after infection; however, neither CDP reduction nor viral replication was inhibited by treatment with 10 mM thymidine. These findings indicate the induction of an RNR activity with an altered allosteric regulation compared to the mouse RNR following MCMV infection and suggest that the virus R1 homologue may complex with the induced cellular R2 protein to reconstitute a new RNR activity.

Animals↗

PubMed1999

The interferon-inducible 204 gene, a member of the Ifi 200 family, is not involved in the antiviral state induction by IFN-alpha, but is required by the mouse cytomegalovirus for its replication.

To examine whether Ifi 200 genes are involved in antiviral state induction by IFNs we expressed mutant forms capable of inactivating the endogenous p204 and analyzed replication of both RNA and DNA viruses following IFN-alpha treatment. Inactivation of p204 does not impair replication of vesicular stomatitis virus, encephalomyocarditis virus, ectromelia virus, and herpes simplex virus 1 and does not alter an IFN-alpha induced antiviral state. By contrast, in cells lacking functional p204, mouse cytomegalovirus (MCMV) replication is strongly inhibited and is not further modulated by IFN-alpha. These results suggest that p204, a member of the Ifi 200 gene family, is not involved in the IFN-alpha-induced antiviral activity against some RNA or DNA viruses, but is required by MCMV for its replication.

3T3 Cells↗

PubMed1999

The HMG protein T160 colocalizes with DNA replication foci and is down-regulated during cell differentiation.

The high mobility group protein T160, the murine homolog of the human structure-specific recognition protein 1, was first supposed to be involved in the process of V-(D)-J recombination, since it could bind to recombination signal sequence probes. We have recently cloned T160 by using an unrelated DNA probe and shown that it binds to either cruciform or linear DNA with no sequence specificity. In this work, we performed a detailed analysis of T160 expression and immunolocalization. We show that T160 is a phosphoprotein broadly conserved from yeast to mammals, with a high level of expression in all the cell lines tested and in tissues containing a high degree of proliferating cells. Indirect immunofluorescence analysis by confocal laser microscopy revealed that T160 distribution in the cell nucleus is not uniform, and focus-like staining was observed. Cell cycle studies by BrdU incorporation suggest that the appearance of T160 nuclear foci is specific of mid to late S phase. Furthermore, while T160 expression does not change during the cell cycle, it is dramatically down-regulated when cells begin to differentiate, as highlighted in C2C12 myoblasts and myotubes. The disappearance of T160 nuclear staining in multinucleated myotubes is shown. Taken together, these data suggest that its function may be less specific than V-(D)-J recombination and more related to some cellular basic process, such as DNA replication or repair.

Amino Acid Sequence↗

PubMed1999

Demonstration of multiple HPV types in laryngeal premalignant lesions using polymerase chain reaction and immunohistochemistry.

Recent evidence has shown that human papillomavirus (HPV) is involved in both the development of carcinoma and in premalignant mucosal lesions of the oral cavity. This study examined the relationship of HPV infection to some pathological features in precancerous lesions of the larynx, not examined extensively so far. Fifty formalin-fixed paraffin-embedded tissue sections containing human laryngeal precancerous lesions were screened for the presence of HPV infection by polymerase chain reaction, and for capsid protein expression by immunohistochemistry with polyclonal antibody directed against the L1 protein. The presence of HPV DNA was detected in 28 of 50 specimens (56%), including 9/12 cases with mild dysplasia (75%), 3/6 cases with moderate dysplasia (50%), and 7/11 cases with severe dysplasia (64%). Multiple HPV infections, containing two or three types, were detected in 17 of the 28 HPV-positive lesions (60%). Of 21 cases with keratosis and no dysplasia, 11 were positive for HPV DNA (52%) and 4 showed L1 staining (36%). By contrast, L1 positivity was revealed only in two lesions with moderate dysplasia, confirming that fully productive HPV infection is strictly dependent on epithelial differentiation and surface keratinization. The probability that HPV is a cofactor in the malignant progression of these lesions is suggested by the fact that 3/4 patients who developed cancer within 50 months were positive for HPV DNA.

Adult↗

PubMed1998

Inhibition of cell proliferation by the interferon-inducible 204 gene, a member of the Ifi 200 cluster.

The role of the IFN-inducible p204 as growth regulator was investigated by transfecting an expression vector constitutively expressing p204 into several cell lines. Like pRB and p107, p204 is a potent growth inhibitor in sensitive cells, as demonstrated by the cell focus assay. Since stable transfectants of sensitive lines constitutively overexpressing p204 could not be established in vitro, we inserted the 204 cDNA into a vector bearing an heavy-metal-inducible promoter. Here we show that proliferation of B6MEF fibroblasts lacking endogenous p204 is strongly inhibited by transient p204 expression in the nucleus. p204 delays G1 progression into the S-phase and cells accumulate with a DNA content equivalent to cells arrested in late G1. Moreover, the role of p204 in the control of cell growth in vivo was investigated by generating transgenic mice in which the Ifi 204 gene was constitutively expressed in all tissues. To this end, expression vectors bearing the 204 cDNA under the control of the SV40 viral promoter were constructed. The overexpression of the p204 transgene achieved by injecting fertilized mouse eggs with these vectors was compatible with embryo development up to the four-cell stage in an in vitro follow-up of 4.5 days. However, no viable animals with an intact copy of the transgene were obtained, suggesting that high and constitutive levels of p204 expression can impair normal embryo development. These findings indicate that p204 plays a negative role in growth regulation and provide new information about the molecular mechanisms exploited by IFNs to inhibit cell proliferation.

Animals↗

PubMed1998

The murine homolog of the HIN 200 family, Ifi 204, is constitutively expressed in myeloid cells and selectively induced in the monocyte/macrophage lineage.

To examine the expression of Ifi 200 genes in vivo and add new information about their function, polyclonal monospecific rabbit antibodies, designated N-term or C-term, were raised against both the N-terminus and C-terminus of the 204 protein (p204) respectively. Western blotting analysis demonstrated that p204 and D3, another member of the Ifi 200 gene family, are constitutively expressed, though at different degrees, in bone marrow, thymus and lymph nodes, and barely detectable in the spleen. Poly rI:rC treatment did not modulate their expression. Peritoneal resident macrophages (Mphi) from untreated mice were negative, but displayed high levels of both p204 and D3 on poly rI:rC treatment. A significant increase of these proteins is also observed when Mphi are cultured overnight in vitro with IFNs or LPS. Lung, kidney and brain were negative for p204 and D3 expression. These results, together with immunohistochemical analysis, demonstrate that the 204 gene has an expression pattern restricted to cells of the myelomonocytic lineage similar to that observed for the human homolog, the myeloid nuclear differentiation antigen (MNDA) suggesting its potential involvement in the differentiation and maturation of this cell lineage.

Animals↗

PubMed1998

Murine cytomegalovirus induces expression and enzyme activity of cellular dihydrofolate reductase in quiescent cells.

Murine cytomegalovirus (MCMV) productively infects quiescent fibroblasts in which the levels of nucleoside triphosphate precursors and cell functions involved in DNA metabolism are minimal. It appears that MCMV has evolved molecular pathways in order to ensure the presence of nucleoside triphosphate precursors for the viral DNA polymerase. Here, we report that MCMV infection of quiescent NIH 3T3 cells markedly stimulates transcription, expression and activity of the cellular dihydrofolate reductase (DHFR), a key enzyme in the synthesis of DNA precursors. DHFR stimulation by MCMV is sensitive to UV irradiation and seems to depend on expression of the viral immediate-early protein pp89. Finally, it has been demonstrated that suppression of virus-induced DHFR activity by the specific inhibitor methotrexate prevents MCMV DNA replication. These observations indicate that induction of host cell DHFR activity by MCMV is required for viral DNA synthesis in quiescent fibroblasts.

3T3 Cells↗

PubMed1997

The high-mobility group protein T160 binds to both linear and cruciform DNA and mediates DNA bending as determined by ring closure.

The high-mobility group protein T160 was isolated by screening a phage library from a murine pre-B-cell line L1210. South-Western experiments have previously shown that this protein binds to V-(D)-J recombination signal sequences, suggesting that it may be a sequence-specific DNA-binding protein. However, neither gel-shift nor footprinting analyses have been successfully employed with the T160 protein, despite an extensive effort. In this study, the T160 protein or truncated forms made soluble through denaturing and renaturing cycles in urea were successfully used in gel-shift experiments showing that T160 binds to cruci-form or linear duplex DNA with no apparent sequence specificity. Furthermore, fragments longer than 100 bp efficiently formed covalently closed circular monomers in the presence of T160 and T4 DNA ligase, indicating that the protein is capable of introducing bends into the duplex. Last, tissue distribution by Western blotting analysis showed that the T160 protein is expressed in various murine tissues in addition to those of lymphoid origin. Considering its broad evolutionary conservation (from plants to mammals) also, these results suggest that the functional role of the T160 protein is not limited to V-(D)-J recombination, but might be involved in basic processes such as DNA replication and repairing, where irregular DNA structures are generated and very likely recognized by HMG domain proteins.

Animals↗

PubMed1997

Molecular cloning and expression of an interferon-inducible protein encoded by gene 203 from the gene 200 cluster.

We report here the complete coding sequence of a 203 cDNA, a member of the interferon-inducible Ifi 200 gene family. By combining reverse-transcriptase PCR and rapid amplification of cDNA ends (RACE) techniques we have obtained a 3.8-kb cDNA corresponding to a 203 mRNA. When used as a probe in northern analysis, its 3' segment hybridized to a 3.8-kb interferon-inducible mRNA, whereas the 5'-end additionally hybridized to a less abundant interferon-inducible 1.8-kb mRNA. Nucleotide sequence analysis revealed that the two mRNAs share the 5'-untranslated region and the same open reading frame, which encodes a hydrophilic protein composed of 408 amino acids. The difference between them is due to a 3'-untranslated region extended by alternative polyadenylation site selection. Furthermore, 203 mRNA was found to be inducible by interferon-alpha in various murine cell lines. Using polyclonal antibodies raised against a segment specific for the 203 protein, we established that p203 protein levels increase on treatment with interferon-alpha in murine fibroblasts and that p203 is located in the nucleus.

3T3 Cells↗

PubMed1997

Decreased expression of the high-mobility group protein T160 by antisense RNA impairs the growth of mouse fibroblasts.

The T160 protein belongs to the HMG-1 box protein family and preferentially binds to non-B-DNA conformations with no sequence specificity. Its exact role has yet to be defined, though it seems to participate in processes involving DNA, such as replication, transcription and recombination. We have used an antisense RNA strategy to investigate its role in cell growth and proliferation. T160 expression is strongly suppressed by stable introduction of an antisense construct into NIH3T3 cells, and this decrease is accompanied by substantial changes in the growth properties of the stable transfectants. Impaired growth of T160- cells was mainly related to two mechanisms: i) decreased rates of cell proliferation at normal serum concentration; and ii) occurrence of cell death by apoptosis at low serum concentration, as demonstrated by both flow cytometry and microscopy. The finding that decreased T160 availability affects cell proliferation, provides further evidence of its involvement in a basic cell function, such as DNA replication.

3T3 Cells↗