Papers by Giuseppe Ragona
Journal of General Virology, 1989
Epstein-Barr virus (EBV) DNA sequences were detected in four established monoblast or early monoc... more Epstein-Barr virus (EBV) DNA sequences were detected in four established monoblast or early monocytic cell lines (CM-S, ROV-S, CV-S and AD-S) obtained from bone marrow of children suffering from maturation defects of haematopoiesis. EBV is present in these cells in a latent state. The viral DNA in these cell lines was analysed by Southern blot hybridization, using a set of cloned EBV DNA fragments from the EBV strain B95-8 as probes. A common spectrum of highly related but distinguishable EBV DNA restriction enzyme sequences was found, suggesting some genomic diversity. Propagation of the cells in long-term culture revealed a gradual decrease of EBV copies per cell in all lines with some minor changes in the restriction pattern of the EBV DNA. These findings demonstrate that human precursor monocyte cells may be susceptible to infection by EBV.
International Journal of Cancer, 1986
Ultrastructural modifications are described in the plasma membrane of in vitro established human ... more Ultrastructural modifications are described in the plasma membrane of in vitro established human B cells. By the freeze-fracture technique, intramembrane particles (IMPs) are quantified in B lymphocytes following Epstein-Barr virus (EBV) transformation in vitro, and in B-lymphoma (Burkitt-type) cells, either positive or negative for EBV genome. Analysis shows an overall increase in IMP density as compared to normal controls. Differences are observed between the protoplasmic and exoplasmic faces of fractured membranes as well as among in vitro transformed and clearly neoplastic cells. Results indicate that conformational changes in IMP distribution parallel neoplastic evolution of transformed cells.
International Journal of Cancer, 1975
Antibody reactivity to Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA) was investigate... more Antibody reactivity to Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA) was investigated by means of the anticomplement immunofluorescence technique on sera from patients with Hodgkin's disease (HD) and from appropriate controls. Antibody levels to other EBV-determined antigens, i.e. viral capsid (VCA) and early antigens (EA), and to measles and rubella viruses, to cytomegalovirus (CMV), and to toxoplasma gondii were also measured. The results of anti-EBV antibody titrations demonstrated that anti-VCA, anti-EA and anti-EBNA reactivity was significantly higher in HD patients than in healthy subjects. There was no significant difference between the distribution of high rubella and measles antibody titers in HD and control sera. The GMT and the incidence of high titer anti-CMV and toxoplasma antibodies were greater in HD patients than in controls. The analysis of the data, according to histological subtypes, showed that the condition of lymphocyte depletion was associated in HD patients with the highest anti-EBNA antibody levels and the lymphocyte predominance with the lowest. This pattern seemed to be peculiar for anti-EBV reactivity, since anti-CMV and anti-toxoplasmn antibody levels in the lymphocytedepleted group of patients did not significantly differ from those of controls. No correlation was found between anti-VCA and anti-EBNA in individual sera of HD patients. This observation suggests that different mechanisms are probably responsible in HD for the release of EBV-related antigens from infected cells.
Journal of Hygiene, 1978
Influenza activity was studied in the Rome population from 1956 to 1976 by analysis of mortality ... more Influenza activity was studied in the Rome population from 1956 to 1976 by analysis of mortality from respiratory causes and from all causes. During cold weather months, type A influenza virus was associated, as a rule, with epidemic excess deaths at two year intervals while type B virus was prevalent twice during isolation data were also compared with epidemic excess mortality during four consecutive years. The evidence obtained indicated that influenza virus isolation alone does not represent a reliable index of epidemic influenza activity in this population. The proportion of deaths attributed to respiratory causes consistently increased in every epidemic, the most pronounced increases occurring during large epidemics. The break-down by age of deaths from respiratory causes in the course of two epidemic periods showed that the percentage distribution of deaths was essentially the same as in non-epidemic periods. This evidence indicates that the same factors influencing the age-related distribution of mortality from respiratory causes during non-epidemic periods, probably affect the fatal outcome of influenza during epidemics.
Journal of Hygiene, 1979
Live attenuated influenza vaccine containing the recombinant of A/Victoria/3/75 with A/PR/8/34 vi... more Live attenuated influenza vaccine containing the recombinant of A/Victoria/3/75 with A/PR/8/34 virus was administered to healthy adults in a field trial aimed at evaluating protection provided by immunization. The study was designed to measure the effect of vaccination on absenteeism from respiratory disease during a natural influenza epidemic. A total of 2115 male employees of the public transport service of Rome volunteered to participate in the trial, 1050 and 1065 receiving vaccine and placebo respectively, in a randomized blind fashion. Vaccination procedure was completed by the end of December 1976. A small-sized outbreak of influenza, due to a viral strain antigenically homologous to the vaccine, occurred during the month of February 1977. Analysis of absenteeism data, classified according to medical certificate, indicated that morbidity from respiratory disease was reduced in vaccinees compared with controls during the epidemic month; the rate of increase of morbidity compared with that of the preceding month was then three times lower in vaccinees than in controls and the difference in absenteeism between the two groups greatly exceeded the ordinary fluctuation that was observed during non-epidemic periods.
New England Journal of Medicine, 1977
We devised a quantitative assay for Epstein-Barr-virus-infected mononuclear leukocytes (virocytes... more We devised a quantitative assay for Epstein-Barr-virus-infected mononuclear leukocytes (virocytes) to determine their prevalence in the blood of patients with acute-phase and convalescent-phase infectious mononucleosis and in healthy Epstein-Barr-virus-seropositive controls. Mononuclear peripheral blood leukocyte suspensions were tested for virus-determined cytoproliferative activity by cocultivation with human cord-cell indicator cultures. The highest levels of virocytes among circulating mononuclear leukocytes were found in the early acute phase of infectious mononucleosis (up to 0.05 per cent). Virocytemia decreased to levels comparable with those of healthy controls (less than 0.00001 per cent) by the third month after onset of infectious mononucleosis. These findings provide a quantitative profile of the course of the infection at cellular level and support existing evidence of the efficiency of immune control mechanisms in limiting Epstein-Barr-virus infection during the course of infectious mononucleosis.
Journal of Clinical Immunology, 1985
Thirteen patients affected with unexplained lymphoadenopathy, fever, weight loss, and diarrhea (l... more Thirteen patients affected with unexplained lymphoadenopathy, fever, weight loss, and diarrhea (lymphoadenopathy syndrome; LAS) were evaluated for the possible appearance of acquired immunodeficiency syndrome (AIDS) and for immunological and virological characterization. The patients belonged to categories of individuals at risk for AIDS and were homosexual and/or drug abusers or hemophiliacs. Lymph node biopsy showed the histological picture of a follicular hyperplasia. The study of cell-mediated immunity (CMI), humoral immune response, and natural killer (NK) activity demonstrated a significant decrease in T cells with the helper/inducer phenotype (OKT 4+ cells) and a relatively increased number of lymphocytes with the suppressor/cytotoxic phenotype (OKT 8+ cells). NK activity was significantly lower than in normal controls. Thein vitro response to policlonal activators (phytohemagglutinin; PHA) and the cutaneous responsiveness to recall skin tests were impaired, whereas immunoglobulin production was increased, mainly in the IgG fraction. Virological studies showed high serum antibody titers to cytomegalovirus (CMV) but a lack of specific CMI as assayed by the leukocyte migration inhibition test (LMIT). CMV was also isolated from the urine specimen of one patient. The antibody pattern to Epstein-Barr virus (EBV) showed the uncommon contemporary presence of both Epstein-Barr nuclear antigen (EBNA) and early antigen (EA) antibodies. Antibodies to human T-lymphotropic retroviruses (HTLV III) were positive in 10 patients and the virus was isolated in 3 of them. In some patients the presence of serum antibodies to HTLV III was not associated with an impairment of the immune function. A group of individuals at risk for AIDS without LAS was also evaluated for the presence of HTLV III antibodies; the percentage of positive sera was 11.4. Nevertheless, individuals without specific antibodies had immunological abnormalities resembling those of LAS HTLV III-positive patients. The possible implications of these findings are discussed.
Journal of Medical Virology, 2001
The presence and variant distribution of human herpesvirus 6 (HHV-6) was investigated by a nested... more The presence and variant distribution of human herpesvirus 6 (HHV-6) was investigated by a nested polymerase chain reaction (PCR) in 118 biopsies from patients affected by nervous tissue tumor (115 primary tumors and 3 metastasis) and in 31 autopsy samples from the brain of healthy individuals. HHV-6 DNA sequences were detected in normal and neoplastic nervous tissue at a frequency of 32% and 37%, respectively. In both tissues, variant A was three times more frequent than the variant B. Peripheral blood lymphocytes (PBLs) derived from seven tumor affected patients contained the same variant as their respective brain sample, as judged by PCR. The expression of HHV-6 encoded immediate early protein p41 was detected by immunohistochemistry in neoplastic but not in normal brain. This may reflect viral reactivation from latency in immunocompromised patients. The seroepidemiological data indicated a frequency distribution of anti-HHV-6 antibodies in patients with brain tumors similar to that found in healthy donors.
Mechanisms of Ageing and Development, 2006
Mutated huntingtin is expressed in nervous and non nervous system included lymphoblasts. Enereget... more Mutated huntingtin is expressed in nervous and non nervous system included lymphoblasts. Eneregetic metabolism is impaired in Huntington's disease (HD) and other neurodegenerative diseases. Human HD lymphoblasts have provided clear-cut data on mitochondnal disruption. Here we report morphological, morphometric and membrane potential differences in mitochondria from lymphoblasts obtained from patients homozygous and heterozygous for the CAG mutation, and controls. Homozygotes, who despite a similar age at onset show a more aggressive phenotype than heterozygotes, had giant mitochondria and a reduced membrane potential. We argue that early mitochondrial impairment at basal level may affect the severity of HD progression in patients.
Cancer Letters, 1995
We report the fractionation of freshly isolated subsets of tonsillar lymphocytes according to cel... more We report the fractionation of freshly isolated subsets of tonsillar lymphocytes according to cell density and double sorting for the differential expression of CD10 and CD77, and their analysis for the presence of Epstein Barr virus genome by nested PCR. All the subsets of tonsillar lymphocytes gave unequivocal evidence for the presence of EBV DNA, when obtained from EBV seropositive individuals. Confirmation of all cases examined demonstrates that B lymphocytes from the germinal centers of tonsils are also involved in carrying the EBV infection in vivo.
Aids Research and Human Retroviruses, 1995
Human herpesvirus 6, a predominantly T lymphotropic virus, has been recently shown to infect some... more Human herpesvirus 6, a predominantly T lymphotropic virus, has been recently shown to infect some EBV-positive B cell lines, and to induce in them the activation of the EBV lytic cycle. Here we have confirmed and extended such observations, showing that (1) this phenomenon is restricted to the variant A of HHV-6: in fact two isolates belonging to the HHV-6 variant B (BA92 and Z29) were neither able to infect any B cell line, independently of the EBV status, nor to induce the EBV genome expression. The only exception is represented by the P3HR1 cells, in which, however, the infection by the variant B does not determine induction of EBV antigens; (2) the presence of the EBV genome contributes to the susceptibility of the B cell lines to HHV-6 infection, increasing the binding sites and the percentage of infectable cells, as detected by immunoelectron microscopy; and (3) HHV-6 infected T cells, transfected with plasmids bearing the promoter regions of the EBV early genes BZLF1 and BMRF1, show a strong transactivation of these promoters.
Cancer Gene Therapy, 2006
Recent studies are reviewed indicating that the transcription factor Egr1 is a direct regulator o... more Recent studies are reviewed indicating that the transcription factor Egr1 is a direct regulator of multiple tumor suppressors including TGFβ1, PTEN, p53 and fibronectin. The downstream pathways of these factors display multiple nodes of interaction with each other suggesting the existence of a functional network of suppressor factors that serves to maintain normal growth regulation and resist the emergence of transformed variants. Paradoxically, Egr-1 is oncogenic in prostate cancer. In the majority of these cancers PTEN and/or p53 is inactive. It is suggested that these defects in the tumor suppressor network allow for the unopposed induction of TGFβ1 and fibronectin, which favor transformation and survival of prostate tumor epithelial cells, explain the role of Egr1 in prostate cancer. Egr1 is a novel and logical target for intervention by gene therapy methods and targeting methods are discussed.
Journal of Neuroimmunology, 2000
Glutamate is the major excitatory neurotransmitter in the mammalian CNS and mediates fast synapti... more Glutamate is the major excitatory neurotransmitter in the mammalian CNS and mediates fast synaptic transmission upon activation of glutamate-gated ion channels. In addition, glutamate modulates a variety of other synaptic responses and intracellular signaling by activating metabotropic glutamate receptors (mGluRs), which are G protein-coupled receptors. The mGluRs are also expressed in nonneuronal tissues and are implicated in a variety of normal biological functions as well as diseases. To study mGluR-activated calcium signaling in neurons, we generated mGluR5 transgenic animals using a Thy1 promoter to drive expression in the forebrain, and one founder unexpectedly developed melanoma. To directly investigate the role of mGluR5 in melanoma formation, we generated mGluR5 transgenic lines under a melanocyte-specific promoter, tyrosinase-related protein 1. A majority of the founders showed a severe phenotype with early onset. Hyperpigmentation of the pinnae and tail could be detected as early as 3-5 d after birth for most of the mGluR5 transgene-positive mice. There was 100% penetrance in the progeny from the tyrosinase-related protein 1-mGluR5 lines generated from founders that developed melanoma. Expression of mGluR5 was detected in melanoma samples by RT-PCR, immunoblotting, and immunohistochemistry. We evaluated the expression of several cancer-related proteins in tumor samples and observed a dramatic increase in the phosphorylation of ERK, implicating ERK as a downstream effector of mGluR5 signaling in tumors. Our findings show that mGluR5-mediated glutamatergic signaling can trigger melanoma in vivo. The aggressive growth and severe phenotype make these mouse lines unique and a potentially powerful tool for therapeutic studies.
Biochemical and Biophysical Research Communications, 2004
Resistance to chemotherapy is a common feature of malignant gliomas. This resistance is mediated ... more 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.
Cancer Cell International, 2004
The aim of this work was to investigate in vitro the putative role of EGR-1 in the growth of glio... more The aim of this work was to investigate in vitro the putative role of EGR-1 in the growth of glioma cells. EGR-1 expression was examined during the early passages in vitro of 17 primary cell lines grown from 3 grade III and from 14 grade IV malignant astrocytoma explants. The explanted tumors were genetically characterized at the p53, MDM2 and INK4a/ARF loci, and fibronectin expression and growth characteristics were examined. A recombinant adenovirus overexpressing EGR-1 was tested in the primary cell lines.
American Journal of Medical Genetics Part B-neuropsychiatric Genetics, 2005
The analysis of somatic CAG triplet variation in lymphoblastoid cell lines from subjects carrying... more The analysis of somatic CAG triplet variation in lymphoblastoid cell lines from subjects carrying alleles of intermediate length (IA33CAG and IA34CAG) in Huntington disease (HD) gene disclosed instability in the DNA of the person, from whom a new expansion mutation of 45 triplets originated. The triplet size increased after about 30 passages in cell cultures in lymphoblasts with the IA34 genotype. Lymphoblasts may provide an appropriate model for studying repeat instability in subjects with poly(CAG) repeat disorders. HD shows somatic, in addition to germ-line instability, highlighting the propensity to somatic CAG variation in human cells even with repeat numbers under the expanded edge. Factors potentially cis acting with the mutation, other than those reported in this study (CCG polymorphic stretch, the deletion of the glutamic acid residue at position 2642 and the 4-codon segment between CAG and CCG polymorphisms), should be searched for and analyzed. © 2004 Wiley-Liss, Inc.
International Journal of Cancer, 1997
Reverse-transcriptase polymerase chain reaction has been used to analyze the expression of 2 late... more Reverse-transcriptase polymerase chain reaction has been used to analyze the expression of 2 latent genes (EBNA-1 and LMP-1) and one replicative gene (BZLF-1) of Epstein-Barr virus in mononuclear cells from bone marrow and peripheral blood of healthy donors. EBV-gene transcription was detected in 8 out of 15 bone-marrow samples. Among these, 5 allowed the detection of latency-associated transcripts in the absence of BZLF-1 expression. Only one sample showed positivity for expression of both latent and lytic genes. In 2 cases, BZLF-1 was the only transcript detected. In peripheral blood, 4 out of 7 samples showed evidence of EBNA-1 transcription; LMP-1 was expressed in 5 samples, and in 2 cases concomitant expression of EBNA-1 and BZLF-1 was detected. These results provide a direct demonstration by RT-PCR of EBV-gene transcription in bone-marrow-resident viral infected cells and suggest, in contrast to previous studies on peripheral blood, that LMP-1 and BZLF-1 are frequently transcribed also in absence of EBV-related disease. The heterogeneous viral gene expression found makes it difficult to define a pattern of viral latency in vivo which coincides with that described for lymphoblastoid or Burkitt's-lymphoma cell lines at different stages of differentiation.
Journal of Medical Virology, 2002
Several reports have suggested an association of human herpesvirus 6 (HHV-6) with multiple sclero... more Several reports have suggested an association of human herpesvirus 6 (HHV-6) with multiple sclerosis. Autoreactive T lymphocytes directed against myelin components seem to contribute to the pathogenesis of the disease. It has been suggested that molecular mimicry between viral and self-antigens might be one of the mechanisms that determine the onset of several autoimmune diseases. Following this hypothesis, the purpose of the present study was to evaluate if HHV-6 could play a role in activating T cells capable of cross-reaction with an important myelin component, the myelin basic protein. T cell lines were established from 22 multiple sclerosis patients and from 16 healthy controls, and their capability to react to both virus and myelin basic protein antigens was compared. The analysis of T cell cross-reactivity in patients and controls did not show significant differences in the HHV-6 ability to activate myelin basic protein-reactive T cells. Similarly, the evaluation of the humoral immune response to HHV-6 in patients and controls did not mirror any abnormality in the HHV-6 status in multiple sclerosis patients. Therefore, although the findings of activity in vitro of T cell lines with dual specificity are consistent with the hypothesis of molecular mimicry, the lack of differences in cross-reactivity between patients and controls do not support molecular mimicry as an important mechanism in the physiopathology of this disease. J. Med. Virol. 68: 268–272, 2002. © 2002 Wiley-Liss, Inc.
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Papers by Giuseppe Ragona