Human monocytic cell lines derived from cord leukocytes by co-cultivation with irradiated CM-S cells.

The CM-S cell line was established from the bone marrow of a child with congenital hypoplastic anemia and resembles its monocyte-macrophage lineage. Lethally x-irradiated CM-S cells from various passages and clones, representing different stages in the progression of the transformed growth phenotype, were tested for their ability to affect the survival and proliferation of normal human cord or adult blood leukocytes in co-culture. One clone, CM-SM, which is tumorigenic in athymic mice, consistently immortalized umbilical cord mononuclear cells but did not immortalize adult peripheral blood leukocytes. Six autonomous monocyte-like diploid cell lines were obtained and all were found to be of cord origin. Three lines were tumorigenic in athymic mice. Attempts to immortalize human leukocytes with cell-free supernatants from CM-S cells were unsuccessful.

The transcription factor Egr1 is a direct regulator of multiple tumor suppressors including TGFbeta1, PTEN, p53, and fibronectin.

Recent studies are reviewed indicating that the transcription factor early growth response-1 (Egr1) is a direct regulator of multiple tumor suppressors including TGFbeta1, 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 serve to maintain normal growth regulation and resist the emergence of transformed variants. Paradoxically, Egr1 is oncogenic in prostate cancer. In the majority of these cancers, PTEN or p53 is inactive. It is suggested that these defects in the suppressor network allow for the unopposed induction of TGFbeta1 and fibronectin, which favor transformation and survival of prostate tumor epithelial cells, and 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.

Expression of Egr-1 correlates with the transformed phenotype and the type of viral latency in EBV genome positive lymphoid cell lines.

In this paper we have investigated the role of Egr-1 in B cell growth regulation by examining the gene expression in a panel of B cell lines, including both EBV genome negative and EBV carrying cell lines. Egr-1 expression correlates with the cellular phenotype and the specific pattern of viral latency established within the individual cell lines. Thus, constitutive activation of Egr-1 gene is invariably associated with unrestricted expression of viral latent genes in all group III EBV genome carrying cell lines. In contrast, Egr-1 expression is abrogated in group I Burkitt tumor cells, irrespective of the EBV genome carrying status. Activated viral gene expression associated with phenotypic conversion of group I cell lines in to group II or III restores the Egr-1 gene expression. Several forms of EGR-1 protein are found within the different groups of cell lines, and the binding activity to DNA consensus sequences was investigated. Finally, time course analysis of Egr-1 expression during the early steps of EBV infection in vitro demonstrated that Egr-1 is upregulated within minutes from the initial interaction with the B lymphocyte.

The early growth response gene EGR-1 behaves as a suppressor gene that is down-regulated independent of ARF/Mdm2 but not p53 alterations in fresh human gliomas.

PURPOSE: EGR-1 is an immediate early gene with diverse functions that include the suppression of growth. EGR-1 is down-regulated many cancer cell types, suggesting a tumor suppressor role, and may critically involve the p53 pathway. The aim of this work was to measure the expression of EGR-1 and the p16/INK4a/ARF-Mdm2-p53 pathway status in fresh human gliomas. EXPERIMENTAL DESIGN: Thirty-one human gliomas with different grades of malignancy were investigated for Egr-1 mRNA and the protein expression, frequency, and spectrum of p53 gene mutations, mdm2 gene amplification, and p16/INK4a/ARF allele loss. RESULTS: The amplification of Mdm2 and the deletion of the p16/INK4a gene was found in 3 and 5 cases, respectively, whereas mutations of p53, including two novel mutations, were observed in 10 other cases. The three types of changes occurred strictly mutually exclusively, emphasizing that these genes operate in a common pathway critical to glioma progression. EGR-1 mRNA was significantly down-regulated in astrocytomas (14.7 +/- 5.1%) and in glioblastomas (33.6 +/- 10.0%) versus normal brain. Overall, EGR-1 mRNA was strongly suppressed (average, 15.2 +/- 13.9%) in 27 of 31 cases (87%), independent of changes in p16/INK4a/ARF and Mdm2; whereas 4 of 31 cases with residual EGR-1 expression as well as the highest EGR-1 variance segregated with p53 mutations. Immunohistochemical analyses confirmed the suppression of EGR-1 protein. CONCLUSIONS: These results indicate that EGR-1 is commonly suppressed in gliomas independent of p16/INK4a/ARF and Mdm2 and that suppression is less crucial in tumors bearing p53 mutations, and these results implicate an EGR-1 growth regulatory mechanism as a target of inactivation during tumor progression.

Expression of metabotropic glutamate receptors in murine thymocytes and thymic stromal cells.

RT-PCR combined with immunoblotting showed the expression of group-I (mGlu1 and 5) and group-II (mGlu2 and 3) metabotropic glutamate receptors in whole mouse thymus, isolated thymocytes and TC-1S thymic stromal cell line. Cytofluorimetric analysis showed that mGlu-5 receptors were absent in CD4(-)/CD8(-) but present in more mature CD4(+) CD8(+) and CD4(+)CD8(-) thymocytes. mGlu-1a receptors showed an opposite pattern of expression with respect to mGlu5, whereas mGlu2/3 receptor expression did not differ between double negative and double positive cells. mGlu receptors expressed in both thymic cell components were functional, as indicated by measurements of polyphosphoinositide hydrolysis or cAMP formation. These data suggest a possible role for mGlu receptor signalling in the thymus.

Epstein-Barr virus DNA is present both in CD10/CD77 positive and negative subsets of human tonsillar lymphocytes.

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.

Human herpesvirus 6 variant A, but not variant B, infects EBV-positive B lymphoid cells, activating the latent EBV genome through a BZLF-1-dependent mechanism.

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.

The transcriptional factor Egr-1 is synthesized by baculovirus-infected insect cells in an active, DNA-binding form.

The Egr-1 (zfp-6) gene encodes a zinc-finger-containing nuclear protein that is rapidly and transiently induced in quiescent cells treated with mitogens. We have constructed baculovirus vectors that synthesize mouse Egr-1 protein initiating at two putative ATG start sites. The ATG site producing the larger protein (Mr, 80,000) is similar, if not identical, to Egr-1 synthesized by serum-stimulated quiescent mouse fibroblasts, thus identifying the likely site for translation. The protein synthesized by the insect cells is active as assayed by its ability to bind to a specific DNA sequence that has been identified as an Egr-1 binding site. The insect cell system will allow further studies of the structure and function of the Egr-1 product, a protein that appears to be an important "master switch" for other genes.

Wilms' tumor gene expression by normal and malignant human B lymphocytes.

Very little is known about Wilms' tumor gene (WT1) expression in B cells and its importance for growth regulation and differentiation. We have investigated WT1 expression in fresh B lymphocytes and in a panel of B-cell lines of normal and malignant origin, including both Epstein-Barr virus (EBV) genome negative and EBV carrying cell lines. WT1 is constitutively activated in all lymphoblastoid cell lines (LCL) derived from EBV immortalization of lymphocytes from normal donors in vitro. These cell lines are distinguished for the presence of activated B-cell markers and an unrestricted expression of viral latent genes. In contrast, WT1 expression is abrogated in normal B lymphocytes and in all Burkitt tumor derived cell lines, irrespective of the EBV genome carrying status and their phenotype pattern. A single step RT-PCR for simultaneous detection of the four spliced transcript isoforms has been applied to confirm their expression. Analysis of variant relative proportions suggested the maintenance of a balanced expression of the isoforms in LCL, as reported in non tumorous tissues. These data, together with the evidence that the replication in vitro of lymphoblastoid cells is not affected by WT1 activation following viral immortalization, support the hypothesis that gene inactivation, in addition to disrupted alternate splicing, may play a role in growth control derangements.

Expression of neurotrophin receptors in normal and malignant B lymphocytes.

In order to define a cellular model suitable for studying, in vitro, the molecular properties and functions of neurotrophin receptors in human lymphocytes, TrkA, TrkB, TrkC and p75(NTR) expression was investigated in a panel of EBV immortalized lymphoblastoid (LCL) and Burkitt lymphoma-derived cell lines (BLs) compared to primary B lymphocytes by RT-PCR and flow cytometric analysis. Our data show that trkA and trkB are transcribed in most B cell lines of normal and malignant origin. For several of them, we also gained first evidence of trkC expression in B cells. All cell lines and primary B cells lack p75(NTR) expression. These data suggest that neurotrophin receptors expression in the B cell lines correlates to some extent with the phenotypic maturation stage and endogenous viral activity levels. Our data suggest that TrkA and TrkB, once activated, provide a partial rescue from apoptosis, whereas TrkC stimulates the progression through the cell cycle without affecting cell survival. Finally, the identification of a number of cell lines showing single expression of one of the Trk receptors has disclosed the availability of a cellular tool for further studies on their function, and mechanisms of signal transduction in the B cell moiety in the absence of p75(NTR).

Imbalance of the Epstein-Barr virus-host relationship in AIDS-related complex patients.

Patients affected by AIDS related-complex (ARC) showed several immunological abnormalities that could lead to a disregulation of immunosurveillance against viral latent infections. We report Epstein-Barr virus (EBV) reactivation was found in seven of eight ARC patients and in two of seven affected by persistent generalized lymphoadenopathy. These patients showed either elevated levels of circulating EBV-positive transformed cells and/or depressed EBV-specific T cell cytotoxicity as assessed by the regression assay. Natural killer cell activity was found to be decreased and correlated with evidence of circulating EBV-infected cells and with impaired EBV-specific immune control. Our data demonstrate that loss of control of EBV latency in the infected host by specific immune mechanisms increases the risk for EBV reactivation and emergence of clones with unlimited growth potential. A role of EBV as a cofactor in the development of ARC is suggested.

Epidemiological evaluation of influenza in Italy.

A statistical study of the mortality from all causes and from respiratory diseases was carried out in an evaluation of the influenza epidemics in Italy from 1956 to 1973. Type A influenzaviruses were responsible for outbreaks every 2-3 years. Type B influenzavirus appeared every 4 years and accounted for a single noteworthy epidemic in 1962-63. From the analysis of data on both general mortality and respiratory mortality a better evaluation of the extent of the influenza outbreaks could be made.

Antibodies to Epstein-Barr virus-associated nuclear antigen and to other viral and non-viral antigens in Hodgkin's disease.

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-toxoplasma antibody levels in the lymphocyte-depleted 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 antigen from infected cells.

Quantitative evaluation of Epstein-Barr-virus-infected mononuclear peripheral blood leukocytes in infectious mononucleosis.

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.

Epstein-Barr virus-related ultrastructural modifications of plasma membrane during B-cell transformation.

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.

Mouse rosette forming cells and surface immunoglobulins in human lymphoid cells.

Peripheral blood lymphocytes from normal subjects were studied for mouse rosette-forming cells (MRFC) and their relationship to surface immunoglobulins (SIg). The majority of MRFC expressed both SIgM and SIgD, although some dissociation between population showing SIgM+/MRFC- and SIgM-/MRFC+ could be seen. A similar pattern of association was found in human derived cultured cells of B lineage, but no simple correlation and the number of SIgM+ cells could be established. Increased percentages of MRFC were detected in two foetal livers but greater dissociation of MRFC and SIgM was shown. This heterogeneity of MRFC may be explained by a selective expression of this surface marker in the early stages of B lymphocyte differentiation.

Seroimmunity to polioviruses in an urban population of Italy.

In a seroepidemiological survey on immunity to polioviruses, carried out in Rome, neutralizing antibodies were titrated on 602 serum samples collected from individuals aged from 6 months to 88 years. The geometric mean titres were 25.24, 25.92, and 18.12 for poliovirus types 1, 2, and 3, respectively. The number of seronegative results for each virus strain was very low (

[Complement fixation, hemagglutination inhibition and neuraminidase inhibition reactions in the seroepidemiologic evaluation of an influenza A epidemic]. / Reazione di fissazione del complemento, emagglutino-inibizione e neuraminidasi-inibizione nella valutazione siero-epidemiologica di un'epidemia da virus influenzale A

Complement-fixing (CF), hemagglutination-inhibiting (HI) and neuraminidase-inhibiting (NI) antibodies in respect of type A influenza virus were titrated on 2080 serum specimens. The sera were collected between December 1974 and May 1975 for the open population of Rome, in groups of an average of 300 samples per month. Influenza type A strains, related to the Port Chalmers variant, were isolated in Rome in January and February 1975, during that time a significant increase of antibody titers and of serum positivity was detected. The increase of positive response for influenza type A antigen was of 21%, 17% and 8% for FC, HI and NI reactions, respectively. The NI reaction proved to be less responsive that the CF and HI reactions for the purposes of sero-epidemiological evaluation of the influenza epidemic.